Materials and methods for treating cancer

ABSTRACT

This document provides methods and materials involved in treating cancer. For example, chimeric antigen receptor T cells having reduced levels of GM-CSF are provided. Also provided as methods for making and using chimeric antigen receptor T cells having reduced levels of GM-CSF.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of PCTInternational Application No. PCT/US19/59275, filed Oct. 31, 2019, whichclaims priority to U.S. Provisional Application No. 62/753,485, filedOct. 31, 2018, which are hereby incorporated by reference.

SEQUENCE LISTING INCORPORATION

The “.txt” Sequence Listing filed with this application by EFS and whichis entitled P-588784-US2-SQL-06JUN21_ST25.txt, is 28.6 kilobytes in sizeand which was created on Jun. 6, 2021, is hereby incorporated byreference.

BACKGROUND 1. Technical Field

This document relates to methods and materials involved in treatingcancer. For example, this document provides methods and materials forusing chimeric antigen receptor T cells having reduced expression levelsof one or more cytokines (e.g., GM-CSF) in an adoptive cell therapy(e.g., a chimeric antigen receptor T cell therapy) to treat a mammal(e.g., a human) having cancer.

2. Background Information

Unprecedented results from pivotal trials evaluating the safety andefficacy of CD19 directed chimeric antigen receptor T cells (CART19)have led to the recent FDA approval of CART19 (Tisagenlecleucel) forrelapsed refractory acute lymphoblastic leukemia (ALL) and CART19(Axi-Cel) for the treatment of diffuse large B cell lymphoma (DLBCL).The application of CART cell therapy is associated with toxicitiesresulting in cytokine release syndrome (CRS) and neurotoxicity.Additionally, the efficacy of CART cell therapy is limited to only 40%durable remissions in lymphoma and 50-60% durable remissions in acuteleukemia. Thus, a critical need exists for compositions and methods forpreventing and treating immunotherapy-related toxicity that occursduring and/or after treatment of cancer with immunotherapeutic methods,such as CAR-T cell therapy.

SUMMARY OF THE INVENTION

This document provides methods and materials for generating T cells(e.g., chimeric antigen receptor (CAR) T cells (CARTs)) having a reducedexpression level of one or more cytokine (e.g., GM-CSF) polypeptides.For example, a T cell (e.g., a CART) can be engineered to have reducedGM-CSF polypeptide expression (e.g., for use in adoptive cell therapy).In some cases, a T cell (e.g., a CART) can be engineered to knock out(KO) a nucleic acid encoding one or more cytokine polypeptides (e.g., aGM-CSF polypeptide) to reduce cytokine polypeptide (e.g., GM-CSFpolypeptide) expression in that T cell. This document also providesmethods and materials for using T cells (e.g., CARTs) having a reducedexpression level of one or more cytokines (e.g., GM-CSF polypeptides).For example, T cells (e.g., CARTs) having a reduced level of GM-CSFpolypeptides can be administered (e.g., in an adoptive cell therapy) toa mammal having cancer to treat the mammal.

In one aspect, this invention provides a method for treating orpreventing CAR-T cell related toxicity in a subject in need thereof, themethod comprising administering to the subject CAR-T cells having aGM-CSF gene inactivation, GM-CSF gene knock-down or gene knockout(GM-CSF^(k/o) CAR-T cells).

In another aspect, this invention provides a method for increasing CAR-Tcell proliferation in a subject treated with GM-CSF-inactivated orGM-CSF^(k/o) CAR-T cells, the method comprising administering to thesubject CAR-T cells having a GM-CSF gene inactivation, GM-CSF geneknock-down or gene knockout (GM-CSF^(k/o) CAR-T cells), whereinadministration of the GM-CSF^(k/o) CAR-T cells increases CAR-Tproliferation in the subject.

In one aspect, this invention provides a method for enhancing anti-tumorefficacy of immunotherapy in a subject, the method comprisingadministering to the subject CAR-T cells having a GM-CSF geneinactivation, GM-CSF gene knock-down or gene knockout (GM-CSF^(k/o)CAR-T cells), wherein administration of these CAR-T cells improves theiranti-tumor efficacy and reduces or prevents immunotherapy-relatedtoxicity.

In another aspect, this invention provides a method for reducing a levelof a non-GM-CSF cytokine in a subject treated with immunotherapy, themethod comprising administering to the subject CAR-T cells having aGM-CSF gene inactivation, GM-CSF gene knock-down or gene knockout(GM-CSF^(k/o) CAR-T cells).

In one aspect, this invention provides a method for GM-CSF geneinactivation, GM-CSF gene knock-down or GM-CSF knockout (KO) in a cellcomprising targeted genome editing or GM-CSF gene silencing.

In one aspect, this invention provides a method for making a chimericantigen receptor T cell having a reduced level of granulocyte-macrophagecolony-stimulating factor (GM-CSF) polypeptides, said method comprising:introducing a nucleic acid construct into an ex vivo T cell, whereinsaid nucleic acid construct comprises: a) a nucleic acid encoding aguide RNA, wherein said guide RNA is complementary to a GM-CSF messengerRNA; b) a nucleic acid encoding a Cas nuclease, and c) a nucleic acidencoding said chimeric antigen receptor.

In another aspect, this invention provides a method for making achimeric antigen receptor T cell having a reduced level ofgranulocyte-macrophage colony-stimulating factor (GM-CSF) polypeptides,said method comprising: introducing a complex into an ex vivo T cell,wherein said complex comprises: a) a guide RNA, wherein said guide RNAis complementary to a GM-CSF messenger RNA; and b) a Cas nuclease; andintroducing a nucleic acid encoding said chimeric antigen receptor intosaid ex vivo T cell.

In an aspect, this invention provides a method for making a chimericantigen receptor T cell having a reduced level of cytokine polypeptides,said method comprising: introducing a nucleic acid construct into an exvivo T cell, wherein said nucleic acid construct comprises: a) a nucleicacid encoding a guide RNA, wherein said guide RNA is complementary to acytokine messenger RNA; b) a nucleic acid encoding a Cas nuclease, andc) a nucleic acid encoding said chimeric antigen receptor.

In another aspect, this invention provides a method for making achimeric antigen receptor T cell having a reduced level of cytokinepolypeptides, said method comprising: introducing a complex into an exvivo T cell, wherein said complex comprises: a) a guide RNA, whereinsaid guide RNA is complementary to a cytokine messenger RNA; and b) aCas nuclease; and introducing a nucleic acid encoding said chimericantigen receptor into said ex vivo T cell.

In a further aspect, this invention provides a method for improving Tcell effector functions of a chimeric antigen receptor T cell, saidmethod comprising: introducing a nucleic acid construct into an ex vivoT cell, wherein said nucleic acid construct comprises: a) a nucleic acidencoding a guide RNA, wherein said guide RNA is complementary to aGM-CSF messenger RNA; b) a nucleic acid encoding a Cas nuclease, and c)a nucleic acid encoding said chimeric antigen receptor.

In an aspect, this invention provides a method for improving T celleffector functions of a chimeric antigen receptor T cell, said methodcomprising: introducing a complex into an ex vivo T cell, wherein saidcomplex comprises: a) a guide RNA, wherein said guide RNA iscomplementary to a GM-CSF messenger RNA; and b) a Cas nuclease; andintroducing a nucleic acid encoding said chimeric antigen receptor intosaid ex vivo T cell.

As demonstrated herein, GM-CSF KO CARTs produce reduced levels of GM-CSFand continue to function normally in both in vitro and in vivo models.Also, as demonstrated herein, GM-CSF KO CARTs can have enhanced CARTcell function and antitumor activity. For example, enhanced CART cellproliferation and anti-tumor activity can be observed after GM-CSFdepletion. CART19 antigen specific proliferation in the presence ofmonocytes can be increased in vitro after GM-CSF depletion. In ALLpatient derived xenografts, CART19 cells can result in a more durabledisease control when combined with lenzilumab, and GM-CSF^(k/o) CARTcells can be more effective in controlling leukemia in NALM6 xenografts.In some cases, GM-CSF KO CARTs can be incorporated into adoptive T celltherapies (e.g., CART cell therapies) to treat, for example, mammalshaving cancer without resulting in CRS and/or neurotoxicity. Forexample, GM-CSF KO CARTs can be incorporated into adoptive T celltherapies (e.g., CART cell therapies) to enhance the therapeutic windowafter CART cell therapy. In some cases, a single construct can be usedboth to introduce a CAR into a cell (e.g., a T cell) and to reduce orknock out expression of one or more cytokine polypeptides in that samecell.

In another aspect, this invention provides a method for treating amammal having cancer, wherein said method comprises administeringchimeric antigen receptor T cells having a reduced level ofgranulocyte-macrophage colony-stimulating factor (GM-CSF) polypeptidesto said mammal.

In still another aspect, this invention provides a method for treating amammal having cancer, wherein said method comprises administeringchimeric antigen receptor T cells having a reduced level of cytokinepolypeptides to said mammal.

In general, one aspect of this document features methods for making aCART cell having a reduced level of cytokine polypeptides. The methodscan include, or consist essentially of introducing a nucleic acidconstruct into an ex vivo T cell, wherein the nucleic acid constructincludes: a) a nucleic acid encoding a guide RNA (gRNA) complementary toa cytokine messenger RNA (mRNA); b) a nucleic acid encoding a Casnuclease, and c) a nucleic acid encoding a chimeric antigen receptor.The cytokine polypeptides can include granulocyte-macrophagecolony-stimulating factor (GM-CSF) polypeptides, interleukin 6 (IL-6)polypeptides, IL-1 polypeptides, M-CSF polypeptides, and/or MIP-1Bpolypeptides. The cytokine polypeptides can be GM-CSF polypeptides, andthe gRNA can include a nucleic acid sequence set forth in SEQ ID NO:1.The Cas nuclease can be a Cas9 nuclease. The nucleic acid encoding theCAR can include a nucleic acid sequence set forth in SEQ ID NO:2. Thenucleic acid construct can be a viral vector (e.g., a lentiviralvector). The CAR can target a tumor-associated antigen. (e.g., CD19).The introducing step can include transduction.

In another aspect, this document features methods for making a CAR Tcell having a reduced level of cytokine polypeptides. The methods caninclude, or consist essentially of, introducing a complex into an exvivo T cell, where the complex includes: a) a gRNA complementary to acytokine mRNA; and b) a Cas nuclease; and introducing a nucleic acidencoding the CAR into the ex vivo T cell. The cytokine polypeptides caninclude GM-CSF Polypeptides and/or IL-6 polypeptides. The cytokinepolypeptides can be GM-CSF polypeptides, and the gRNA can include anucleic acid sequence set forth in SEQ ID NO:1. The Cas nuclease can bea Cas9 nuclease. The nucleic acid encoding the CAR can include a nucleicacid sequence set forth in SEQ ID NO:2. The complex can be aribonucleoprotein (RNP). The CAR can target a tumor-associated antigen(e.g., CD19). The introducing steps can include electroporation.

In another aspect, this document features methods for making a CAR Tcell having a reduced level of GM-CSF polypeptides. The methods caninclude, or consist essentially of introducing a nucleic acid constructinto an ex vivo T cell, where the nucleic acid construct includes: a) anucleic acid encoding a gRNA complementary to a GM-CSF mRNA; b) anucleic acid encoding a Cas nuclease, and c) a nucleic acid encoding theCAR. The gRNA can include a nucleic acid sequence set forth in SEQ IDNO:1. The Cas nuclease can be a Cas9 nuclease. The nucleic acid encodingthe CAR can include a nucleic acid sequence set forth in SEQ ID NO:2.The nucleic acid construct can be a viral vector (e.g., a lentiviralvector). The CAR can target a tumor-associated antigen (e.g., CD19). Theintroducing step can include transduction.

In another aspect, this document features methods for making a CAR Tcell having a reduced level of GM-CSF polypeptides. The methods caninclude, or consist essentially of, introducing a complex into an exvivo T cell, where the complex includes: a) a gRNA complementary to aGM-CSF mRNA; and b) a Cas nuclease; and introducing a nucleic acidencoding the CAR into the ex vivo T cell. The gRNA can include a nucleicacid sequence set forth in SEQ ID NO:1. The Cas nuclease can be a Cas9nuclease. The nucleic acid encoding the CAR can include a nucleic acidsequence set forth in SEQ ID NO:2. The complex can be a RNP. The CAR cantarget a tumor-associated antigen (e.g., CD19). The introducing stepscan include electroporation.

In another aspect, this document features methods for treating a mammalhaving cancer. The methods can include, or consist essentially of,administering CART cells having a reduced level of cytokine polypeptidesto a mammal having cancer. The cytokine polypeptides can include GM-CSFpolypeptides and/or IL-6 polypeptides. The cytokine polypeptides can beGM-CSF polypeptides, and the gRNA can include a nucleic acid sequenceset forth in SEQ ID NO:1. The mammal can be a human. The cancer can be alymphoma (e.g., a DLBCL). The cancer can be a leukemia (e.g., an ALL).The CAR can target a tumor-associated antigen (e.g., CD19).

In another aspect, this document features methods for treating a mammalhaving cancer. The methods can include, or consist essentially of,administering CAR T cells having a reduced level of GM-CSF polypeptidesto a mammal having cancer. The mammal can be a human. The cancer can bea lymphoma (e.g., a DLBCL). The cancer can be a leukemia (e.g., ALL).The cancer can be mantle cell lymphoma. The cancer can be follicularlymphoma. The cancer can be multiple myeloma. The CAR can target atumor-associated antigen (e.g., CD19 or B-cell maturation antigen(BCMA)).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription, examples, and drawings, and from the claims. It should beunderstood, however, that the detailed description and specific exampleswhile indicating certain embodiments of the invention are given by wayof illustration only, since various changes and modifications within thespirit and scope of the invention will become apparent to those skilledin the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure, the inventions of which can be better understood byreference to one or more of these drawings in combination with thedetailed description of specific embodiments presented herein. Thepatent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 contains a schematic of an exemplary method of using CRISPR toengineer a GM-CSF knock out (KO) cell. Guide RNA (GACCTGCCTACAGACCCGCC;SEQ ID NO:1) targeting exon 3 of GM-CSF (also known ascolony-stimulating factor 2 (CSF2)) was synthesized and cloned into alentivirus (LV) plasmid. This LV plasmid was used to transduce 293Tcells and lentivirus particles were collected at 24 hours and 48 hoursand were concentrated. To generate GM-CSF knocked out CART cells, Tcells were stimulated with CD3/CD28 beads on day 0. On day 1, T cellswere transduced with CAR19 lentivirus particles, and simultaneously withGMCSF knockout CRISPR/Cas9 lentivirus particles. T cells were expandedfor 8 days and then harvested.

FIGS. 2A-2B show CAR transduction and GM-CSF knockout efficiency. FIG.2A contains a graph showing that CRISPR/Cas9 lentivirus with a guide RNAdirected to exon 3 of GM-CSF resulted in a knockout efficiency of 24.1%.At the end of the expansion, CART cells were harvested, and DNA wasisolated and sent for sequencing to be compared to control sequences.This yielded in a knockout efficiency of 24.1%. FIG. 2B contains a flowcytometric analysis showing that CAR transduction efficiency aftertransduction with lentivirus was 73%. Flow cytometric analysis wasperformed on Day 6 after lentivirus transduction.

FIG. 3 shows that GM-CSF KO CART19 cells produce less GM-CSF compared toCART cells, and GM-CSF knockout control T cells produce less amount ofGM-CSF compared to control untransduced T cells (UTD). CART19, GM-CSF KOCART19, UTD, or GM-CSF KO UTD were co-cultured with the CD19 positivecell line NALM6 at a ratio of 1:5. 4 hours later, the cells wereharvested, permeabilized, and fixed; and intra-cellular staining forcytokines was performed.

FIG. 4 shows that GM-CSF KO CART19 cells expand more robustly comparedto CART19. After T cells were transduced with the virus, their expansionkinetics was followed. GM-CSF KO expand more robustly compared to CART19alone.

FIG. 5 shows an exemplary nucleic acid sequence (SEQ ID NO:2) encoding aCAR targeting CD19 (CAR19).

FIGS. 6A-6D show that GM-CSF neutralization in vitro enhances CAR-T cellproliferation in the presence of monocytes and does not impair CAR-Tcell effector function. FIG. 6A contains a graph showing that lenzilumabneutralizes CAR-T cell produced GM-CSF in vitro compared to isotypecontrol treatment as assayed by multiplex after 3 days of culture withCART19 in media alone or CART19 co-cultured with NALM6, n=2 experiments,2 replicates per experiment, representative experiment depicted,***p<0.001 between lenzilumab and isotype control treatment, t test,mean±SEM. FIG. 6B contains a graph showing that GM-CSF neutralizingantibody treatment did not inhibit the ability of CAR-T cells toproliferate as assayed by CSFE flow cytometry proliferation assay oflive CD3 cells, n=2 experiments, 2 replicates per experiment,representative experiment at 3 day time point depicted, ns p>0.05between lenzilumab and isotype control treatment, t test, mean±SEM.Alone: CART19 in media alone, MOLM13: CART19+MOLM13, PMA/ION: CART19+5ng/mL PMA/0.1 μg/mL ION, NALM6: CART19+NALM6. FIG. 6C contains a graphshowing that lenzilumab enhanced the proliferation of CART19 compared toisotype control treated with CART19 when co-cultured with monocytes n=3biologic replicates at 3 day time point, 2 replicates per biologicalreplicate, ****p<0.0001, mean±SEM. FIG. 6D contains a graph showing thatlenzilumab treatment did not inhibit cytotoxicity of CART19 oruntransduced T cells (UTD) when cultured with NALM6, n=2 experiments, 2replicates per experiment, representative experiment at 48 hr time pointdepicted, ns p>0.05 between lenzilumab and isotype control treatment, ttest, mean±SEM.

FIGS. 7A-7E show that GM-CSF neutralization in vivo enhances CAR-T cellanti-tumor activity in xenograft models. FIG. 7A contains anexperimental schema showing that NSG mice were injected with the CD19+luciferase+ cell line NALM6 (1×10⁶ cells per mouse I.V). 4-6 days later,mice were imaged, randomized, and received 1-1.5×10⁶CAR-T19 orequivalent number of total cells of control UTD cells the following daywith either lenzilumab or control IgG (10 mg/Kg, given IP daily for 10days, starting on the day of CAR-T injection). Mice were followed withserial bioluminescence imaging to assess disease burden beginning day 7post CAR-T cell injection and were followed for overall survival. Tailvein bleeding was performed 7-8 days after CAR-T cell injection. FIG. 7Bcontains a graph showing that lenzilumab neutralizes CAR-T producedserum GM-CSF in vivo compared to isotype control treatment as assayed byGM-CSF singleplex, n=2 experiments, 7-8 mice per group, representativeexperiment, serum from day 8 post CAR-T cell/UTD injection, ***p<0.001between lenzilumab and isotype control treatment, t test, mean±SEM. FIG.7C contains a graph showing that lenzilumab treated CAR-T in vivo areequally effective at controlling tumor burden compared to isotypecontrol treated CAR-T in a high tumor burden relapse xenograft model ofALL, day 7 post CAR-T injection, n=2 experiments, 7-8 mice per group,representative experiment depicted, ***p<0.001, *p<0.05, ns p>0.05, ttest, mean±SEM. FIG. 7D contains an experimental schema showing that NSGmice were injected with the blasts derived from patients with ALL (1×10⁶cells per mouse I.V). Mice were bled serially and when the CD19+ cells≥1/uL, mice were randomized to receive 5×10⁶ CART19 (transductionefficiency is around 50%) or UTD cells with either lenzilumab or controlIgG (10 mg/Kg, given IP daily for 10 days, starting on the day of CAR-Tinjection). Mice were followed with serial tail vein bleeding to assessdisease burden beginning day 14 post CAR-T cell injection and werefollowed for overall survival. FIG. 7E contains a graph showing thatlenzilumab treatment with CAR-T therapy results in more sustainedcontrol of tumor burden over time in a primary acute lymphoblasticleukemia (ALL) xenograft model compared to isotype control treatmentwith CAR-T therapy, 6 mice per group, **p<0.01, *p<0.05, ns p>0.05, ttest, mean±SEM.

FIG. 8 contains a graph showing that lenzilumab+CAR-T cell treated micehave comparable survival compared to isotype control+CAR-T cell treatedmice in a high tumor burden relapse xenograft model of ALL. n=2experiments, 7-8 mice per group, representative experiment depicted,****p<0.0001, ***p<0.001, *p<0.05, log-rank.

FIG. 9 contains a graph showing a representative TIDE sequence to verifygenome alteration in the GM-CSF CRISPR Cas9 knockout CAR-T cells. n=2experiments, representative experiment depicted.

FIGS. 10A-10E show that GM-CSF CRISPR knockout CAR-T cells exhibitreduced expression of GM-CSF, similar levels of key cytokines, andenhanced anti-tumor activity. FIG. 10A contains graphs showing that theCRISPR Cas9 GM-CSF^(k/o) CAR-T exhibit reduced GMCSF production comparedto wild type CART19, but other cytokine production and degranulation arenot inhibited by the GM-CSF gene disruption, n=3 experiments, 2replicates per experiment, ***p<0.001, *p<0.05, ns p>0.05 comparingGM-CSF k/o CAR-T and CAR-T, t test, mean±SEM.

FIG. 10B contains a graph showing that GM-CSF k/o CAR-T have reducedserum human GM-CSF in vivo compared to CAR-T treatment as assayed bymultiplex, 5-6 mice per group (4-6 at time of bleed, 8 days post CARTinjection), ****p<0.0001, ***p<0.001 between GM-CSF k/o CAR-T cells andwild type CAR-T cells, t test, mean±SEM. FIG. 10C contains a graphshowing that GM-CSF^(k/o) CART19 in vivo enhances overall survivalcompared to wild type CART19 in a high tumor burden relapse xenograftmodel of ALL, 5-6 mice per group, **p<0.01, log-rank. FIGS. 10D and 10Econtain heat maps showing human (FIG. 10D) and mouse (FIG. 10E)cytokines from multiplex of serum, other than human GM-CSF, show nostatistical differences between the GM-CSF k/o CAR-T cells and wild typeCAR-T cells, further implicating critical T-cell cytokines aren'tadversely depleted by reducing GM-CSF expression, 5-6 mice per group(4-6 at time of bleed), ****p<0.0001, t test.

FIG. 11 contains a graph showing that GM-CSF knockout CAR-T cells invivo shows slightly enhanced control of tumor burden compared to CAR-Tina high tumor burden relapse xenograft model of ALL. Days post CAR-Tinjection listed on x-axis, 5-6 mice per group (2 remained in UTD groupat day 13), representative experiment depicted, ****p<0.0001, *p<0.05, 2way ANOVA, mean±SEM.

FIGS. 12A-12D show that patient derived xenograft model forneurotoxicity and cytokine release syndrome. FIG. 12A contains anexperimental schema showing that mice received 1-3×10⁶ primary blastsderived from the peripheral blood of patients with primary ALL. Micewere monitored for engraftment for 10-13 weeks via tail vein bleeding.When serum CD19+ cells were 2:10 cells/uL the mice received CART19(2-5×10⁶ cells) and commenced antibody therapy for a total of 10 days,as indicated. Mice were weighed on a daily basis as a measure of theirwell-being. Mouse brain MRIs were performed 5-6 days post CART19injection and tail vein bleeding for cytokine and T cell analysis wasperformed 4-11 days post CART19 injection, 2 independent experiments.FIG. 12B contains a graph showing that combination of GM-CSFneutralization with CART19 is equally effective as isotype controlantibodies combined with CART19 in controlling CD19+ burden of ALLcells, representative experiment, 3 mice per group, 11 days post CART19injection, *p<0.05 between GM-CSF neutralization+CART19 and isotypecontrol+CART19, t test, mean±SEM. FIG. 12C contains an image showingthat brain MRI with CART19 therapy exhibits T1 enhancement, suggestiveof brain blood-brain barrier disruption and possible edema. 3 mice pergroup, 5-6 days post CART19 injection, representative image. FIG. 12Dcontains graphs showing that high tumor burden primary ALL xenograftstreated with CART19 show human CD3 cell infiltration of the braincompared to untreated PDX controls. 3 mice per group, representativeimage.

FIGS. 13A-13D show that GM-CSF neutralization in vivo amelioratescytokine release syndrome after CART19 therapy in a xenograft model.FIG. 13A contains a graph showing that lenzilumab and anti-mouse GM-CSFantibody prevent CRS induced weight loss compared to mice treated withCART19 and isotype control antibodies, 3 mice per group, 2 way anova,mean±SEM. FIG. 13B contains a graph showing that human GM-CSF wasneutralized in patient derived xenografts treated with lenzilumab andmouse GM-CSF neutralizing antibody, 3 mice per group, ***p<0.001,*p<0.05, t test, mean±SEM. FIG. 13C contains a heat map showing thathuman cytokines (serum collected 11 days after CART19 injection) exhibitincrease in cytokines typical of CRS after CART19 treatment. GM-CSFneutralization results in significant decrease in several cytokinescompared to mice treated with CART19 and isotype control antibodies,including several myeloid associated cytokines, as indicated in thepanel, 3 mice per group, serum from day 11 post CART19 injection,***p<0.001, **p<0.01, *p<0.05, comparing GM-CSF neutralizing antibodytreated and isotype control treated mice that received CAR-T celltherapy, t test. FIG. 13D contains a heat map showing that mousecytokines (serum collected 11 days after CART19 injection) exhibitincrease in mouse cytokines typical of CRS after CART19 treatment.GM-CSF neutralization results in significant decrease in severalcytokines compared to treated with CART19 with control antibodies,including several myeloid differentiating cytokines, as indicated in thepanel, 3 mice per group, serum from day 11 post CART19 injection,*p<0.05, comparing GM-CSF neutralizing antibody treated and isotypecontrol treated mice that received CAR-T cell therapy, t test.

FIGS. 14A-14D show that GM-CSF neutralization in vivo amelioratesneurotoxicity after CART19 therapy in a xenograft model. FIGS. 14A and14B show that gadolinium enhanced T1-hyperintensity (cubic mm) MRIshowed that GM-CSF neutralization helped reduced brain inflammation,blood-brain barrier disruption, and possible edema compared to isotypecontrol (A) representative images, (B) 3 mice per group, **p<0.01,*p<0.05, 1 way ANOVA, mean±SD. FIG. 14C contains a graph showing thathuman CD3 T cells were present in the brain after treatment with CART19therapy. GM-CSF neutralization resulted in a trend toward decreased CD3infiltration in the brain as assayed by flow cytometry in brainhemispheres, 3 mice per group, mean±SEM. FIG. 14D contains a graphshowing that CD11b+ bright macrophages were decreased in the brains ofmice receiving GM-CSF neutralization during CAR-T therapy compared toisotype control during CAR-T therapy as assayed by flow cytometry inbrain hemispheres, 3 mice per group, mean±SEM.

FIGS. 15A-15B show an exemplary generation of GM-CSFk/o CART19 cells.The experimental schema depicts the schema (FIG. 15A), gRNA sequence(FIG. 15B), and primer sequences (FIG. 15B) for generation of GM-CSFk/oCART19. To generate GM-CSFk/o CART19 cells, gRNA was clones into a Cas9lentivirus vector under the control of a U6 promotor and used forlentivirus production. T cells derived from normal donors werestimulated with CD3/CD28 beads and dual transduced with CAR19 virus andCRISPR/Cas9 virus 24 hours later. CD3/CD28 magnetic bead removal wasperformed on Day +6 and GM-CSFk/o CART19 cells or control CART19 cellswere cryopreserved on Day 8.

FIG. 16 shows a flow chart for procedures used in RNA sequencing. Thebinary base call data was converted to fastq using Illumina bcl2fastqsoftware. The adapter sequences were removed using Trimmomatic, andFastQC was used to check for quality. The latest human (GRCh38) andmouse (GRCm38) reference genomes were downloaded from NCBI. Genome indexfiles were generated using STAR30, and the paired end reads were mappedto the genome for each condition. HTSeq was used to generate expressioncounts for each gene, and DeSeq2 was used to calculate differentialexpression. Gene ontology was assessed using Enrichr.

FIGS. 17A-17B show respectively, that CD14+ cells are a greaterproportion of the CNS cell population in human patients with grade 3 orabove neurotoxicity (FIG. 17A) and that anti-hGM-CF antibody,Lenzilumab, caused a reduction in CNS infiltration by CD14+ cells and byCD11b+ cells in the primary ALL mouse model used for the NT experiments(FIG. 17B), as detailed in Example 4.

FIGS. 18A-18E show GM-CSF knockout via CRISPR/Cas9 does not impairCART19 production and effector functions. A-B) CRISPR/Cas9 depletion ofGM-CSF in CART19 cells generated little to no GM-CSF upon CAR19stimulation. Representative flow plot (FIG. 18A) or bar graph (FIG. 18B)showing the levels of GM-CSF detected on live CD3+ cells byintracellular flow cytometric staining upon stimulation with CD19+ cellline Nalm6 (one-way ANOVA, p<0.0001; 4 biological replicates, 2technical replicates) FIG. 18C shows CAR19 expression is not impaired bydepletion of GM-CSF via CRISPR/Cas9. Representative flow plot showing nodifferences in CAR19 expression between GM-CSF^(WT) vs GM-CSF^(KO)CART19 cells after CAR19 stimulation via flow cytometric staining. FIG.18D shows GM-CSF disruption does not affect the composition of CART19(CD4:CD8 ratio) at rest or upon activation. UTD, GM-CSF^(WT) orGM-CSF^(KO) CART19 cells were co-cultured with either CD19+ cell lineNalm6 (CAR stimulation), CD38/CD28 beads (TCR stimulation) orPMA/Ionomycin (Ca+ influx stimulation) for 5 days, followed by flowcytometric staining of CD4 and CD8 staining (one-way ANOVA, ns=notsignificant; 2 biological replicates, 2 technical replicates). FIG. 18Eshows that CSF^(KO) CART19 show enhanced delayed proliferation.GM-CSF^(KO) and GM-CSF^(WT) CART19 cells were co-cultured withirradiated CD19+ cell line Nalm6, and cell counts were obtained dailyfor 6 days (one-way ANOVA, *p<0.05; 2 biological replicates).

FIGS. 19A-19G show CRISPR/Cas9 editing of GM-CSF in CART cells isprecise and specific. FIG. 19A shows there is not a significantdifference in number of single nucleotide variants (SNVs) or insertionsand deletions (indels) between GM-CSF^(KO) conditions and controls.Analysis of single nucleotide polymorphisms by whole exome sequencing onthree biological replicates (n.s.=not significant; Wilcoxon signed ranktest). FIG. 19B shows CSF2 gene-specific editing is precise. Insertionor deletion of cytosine at base pair 132074828 is the only SNV or indelidentified in chromosome 5 (CSF2, exon 3) on three biologicalreplicates. FIG. 19C shows potential off targets predicted by availabletools are not edited. CCTop predicted targets in exonic regions. Onlyedit found in our dataset is CSF2 (CRISPRater score: 0.743377). FIGS.19D-19E show GM-CSF receptors (a and P subunits) are activated upon of Tcell and CART19 cells expansion. Untransduced (UTD) T cells wereisolated from peripheral blood mononuclear cells (PBMCS) and stimulatedover a 6-day expansion period with CD3/CD28 beads. Flow cytometricanalysis was performed in order to assess GM-CSF2R a and P subunitsexpression at days 0, 2, 4 and 6 (Scatter plot (FIG. 19D) andrepresentative flow plot (FIG. 19E), 2 biological replicates). FIGS.19F-19G show GM-CSF2R a and P subunits are upregulated in activatedGM-CSF^(WT) CART19 or GM-CSF^(KO) CART19. GM-CSF^(KO) and GM-CSF^(WT)CART19 cells were activated with either CD3/CD28 beads for 6 days (FIG.19F) or CD19+ cell line Nalm6 for 24 hours (FIG. 19G). Flow cytometricanalysis was performed in order to assess the expression of GM-CSF2R aand P subunits expression on gated on live CD3 (two-way ANOVA;**p-value<0.01, ***p<0.001, ****p<0.0001).

FIGS. 20A-20F show CRISPR/Cas9-mediated depletion of GM-CSF in CARTcells results in decreased T cell apoptosis and AICD. FIG. 20A showsGM-CSF^(WT) CART19 cells are more apoptotic when stimulated through theCAR (CD19+ Nalm6) than TCR (CD3/CD28 beads). CART19 cells wereco-cultured with CD19+ cell line Nalm6 (CAR stimulation), CD38/CD28beads (TCR stimulation) and PMA/Ionomycin (Ca+ influx stimulation). Flowcytometric staining for Annexin V, 7-AAD, and CD3 was performed at 0 hrand 2 hr (two-way ANOVA; **p-value<0.01, ***p<0.001, ****p<0.0001; 4biological replicates). FIG. 20B shows representative flow plot ofshowing the expression of GM-CSF^(WT) CART19 showing apoptotic cells(Annexin V+, 7-AAD-). GM-CSF^(WT) CART19 cells were co-cultured withCD19+ Nalm6. FIGS. 20C-20D show GM-CSF^(KO) CART19 cells are lessapoptotic than GM-CSF^(WT)CART19 cells upon stimulation via CAR ornon-specifically. GM-CSF^(KO) CART19 and GM-CSF^(WT) CART19 wereco-cultured with the CD19+ cell line Nalm6 (FIG. 20C) or PMA/Ionomycin(FIG. 20D). Flow cytometric analysis was performed in order to measureapoptotic cells (Annexin+, 7-AAD-) at 0 hr, 1 hr, 2 hr and 4 hr (two-wayANOVA; **p-value<0.01, ***p<0.001, ****p<0.0001; 4 biologicalreplicates, 3 technical replicates). FIGS. 20E-20F show GM-CSFdisruption ameliorates CART cell apoptosis. GM-CSF^(WT)CART19 andGM-CSF^(KO) CART19 cells were cultured in the presence of irradiatedCD19+ cell line Nalm6 for 0 hr, 2 hr, or 6 hr. TUNEL assay via flowcytometry was performed at each time point in order to measure apoptosisbased on bromolated deoxyuridine triphosphate nucleotide (BrdU) levelson fragmentated DNA on CART cells (two-way ANOVA; ns=not significant; 2biological replicates). Bar plot (FIG. 20E) and representative flow plot(FIG. 20F) of % apoptosis by BrdU of G0-G1 phase.

FIGS. 21A-21E show GM-CSF^(KO) CART19 cells exhibit a distincttranscriptomic profile in comparison to GM-CSF^(WT) CART19 cells. A-C)Comparison of gene expression between untransduced T cells (UTD), CART19cells and GM-CSF^(KO) CART19 cells via RNA-seq. Differential expressionby heatmap (FIG. 21A), volcano plot (FIG. 21B), or principal componentanalysis (FIG. 21C) on RNA isolated from untransduced T cells (UTD),CART19, and GM-CSF^(KO)CART19 cells on day 8 of CART expansion (adj.p-value<0.05, 3 biological replicates). FIG. 21D shows apoptoticpathways are enriched in GM-CSF^(KO) CART19. Gene set enrichmentanalysis of significantly downregulated genes using Enrichr(p-value<0.05). FIG. 21E shows apoptosis is not impaired on CART19 cellsthat were produced in the presence of anti-GM-CSF antibody. CART19 orCART19 cells generated in the presence of anti-GM-CSF antibody wereco-cultured in the presence of CD19+ cell line Nalm6 and flow cytometricstaining for Annexin V, 7-AAD, and CD3 was performed at 0 hr, 1 hr, 2 hr4 hr, and 4 hr (two-way ANOVA; ns=not significant; 3 biologicalreplicates, 3 technical replicates).

FIGS. 22A-22N show GM-CSF disruption on CART19 modulates its earlyactivation and anti-tumor activity. FIGS. 22A-22H show GM-CSF^(KO)CART19 cells showed altered expression levels of T cell activationmarkers. GM-CSF^(KO) or GM-CSF^(WT) CART19 cells were co-cultured withCD19+ Nalm6 for 24 hrs and flow cytometric staining is performed inorder to measure CD3 (FIG. 22A), CD45 (FIG. 22B), CD69 (FIGS. 22C and22D), HLA-DR (FIGS. 22E and 22F) and CD25 (FIGS. 22G and 22H) (one-wayANOVA; **p<0.01, ***p<0.001, ****p<0.0001; 2 biological replicates, 2technical replicates). FIGS. 22I-22L show GM-CSF disruption reducesearly CART cell activation and shows prolonged expansion in an in vivoJeKo-1 relapse xenograft model. Experimental schema showing NSG miceengrafted with the CD19+ luciferase+ cell line JeKo-1 (1×10⁶ cellsintravenous [i.v.] and randomized to treatment with UTD T cells,GM-CSF^(WT)CART19 cells, and GM-CSF^(KO) CART19 cells (1×10⁶ cells i.v.)(FIG. 22I). Tumor burden by bioluminescent imaging over 20 days afterCART cell therapy (two-way ANOVA; ***p<0.001) (FIG. 22J). FIG. 22K showsGM-CSF^(KO) CART19 cells reduced activation in vivo. Peripheral bloodanalysis of UTD, GM-CSF^(WT) CART19 and GM-CSF^(KO) CART19 cells whereCD69, HLA-DR, and CD25 were measured by flow cytometry (one-way ANOVA,*p<0.05). FIG. 22L shows GM-CSF^(KO) CART19 cells exhibit enhanceddelayed proliferation in vivo. Peripheral blood analysis of UTD,GM-CSF^(WT) CART19 and GM-CSF^(KO) CART19 cells where CD3+ cells werequantified (one-way ANOVA, ***p<0.001). FIG. 22M shows GM-CSF^(KO)CART19cells exhibit prolonged survival in vivo. FIG. 22N shows GM-CSF^(KO)CART19 cells exhibit reduced expression of TRAIL-R1. GM-CSF^(WT) andGM-CSF^(KO) CART19 are co-cultured with CD19+ Nalm6 for 24 hours andflow cytometric staining is performed (one-way ANOVA, ****<0.0001; 2biological replicates).

FIGS. 23A-23D show reduced CART19 cell apoptosis following GM-CSFdisruption is due to modulation of intrinsic, and not extrinsic,apoptosis pathways. FIGS. 23A-23B show there is no difference inapoptotic levels between GM-CSF^(WT) CART19 or GM-CSF^(KO) CART19 whendeath receptors Fas or TRAIL-R2 (DR5) are blocked. GM-CSF^(WT) CART19 orGM-CSF^(KO)CART19 cells were co-cultured with CD19+ cell line Nalm6 inthe presence of either an IgG Isotype control or a monoclonal antibodyagainst Fas or TRAIL-R2 (10 ng/mL). Flow cytometric staining for AnnexinV, 7-AAD, and CD3 was performed after 24 hours (two-way ANOVA; **p-value<0.01, ***p<0.001, ****p<0.0001; 3 biological replicates, 2technical replicates). FIGS. 23C-23D show GM-CSF disruption amelioratesCART cell apoptosis through modulation of intrinsic pathways.GM-CSF^(WT) CART19 and GM-CSF^(KO) CART19 cells were co-cultured withCD19+ Nalm6 and western blot for BID was performed at 0 hr, 2 hr, 4 hrand 6 hr (two-way ANOVA; **p-value<0.01, ***p<0.001, ****p<0.0001; 2biological replicates).

FIGS. 24A-24C show a schematic of GM-CSF^(KO) CART19 production with aCRISPR/Cas9 lentiviral vector, similarity of GM-CSF^(KO) CART19 cellsand GM-CSF^(WT)CART19 cells in killing assay and a schematic of CART19production in the presence of GM-CSF blocking antibody. FIG. 24A showsschema of GM-CSF^(KO) CART19 production with CRISPR/Cas9 lentiviralvector. FIG. 24B shows GM-CSF^(WT)CART19 or GM-CSF^(KO) CART19 cells donot show a difference in killing (two-way ANOVA; ns=not significant).FIG. 24C shows schema of CART19 production in the presence of GM-CSFblocking antibody.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description which forms a part of this disclosure. Itis to be understood that this invention is not limited to the specificmethods, products, conditions or parameters described and/or shownherein, and that the terminology used herein is for the purpose ofdescribing particular embodiments by way of example only and is notintended to be limiting of the claimed invention.

As employed above and throughout the disclosure, the following terms andabbreviations, unless otherwise indicated, shall be understood to havethe following meanings.

In this disclosure the singular forms “a,” “an,” and “the” include theplural reference, and reference to a particular numerical value includesat least that particular value, unless the context clearly indicatesotherwise. Thus, for example, a reference to “a compound” is a referenceto one or more of such compounds and equivalents thereof known to thoseskilled in the art, and so forth. The term “plurality,” as used herein,means more than one. When a range of values is expressed, anotherembodiment includes from the one particular and/or to the otherparticular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it is understood thatthe particular value forms another embodiment. All ranges are inclusiveand combinable.

As used herein, the terms “component,” “composition,” “composition ofcompounds,” “compound,” “drug,” “pharmacologically active agent,”“active agent,” “therapeutic,” “therapy,” “treatment,” or “medicament”are used interchangeably herein to refer to a compound or compounds orcomposition of matter which, when administered to a subject (human oranimal) induces a desired pharmacological and/or physiologic effect bylocal and/or systemic action.

As used herein, the terms “treatment” or “therapy” (as well as differentforms thereof) include preventative (e.g., prophylactic), curative orpalliative treatment. As used herein, the term “treating” includesalleviating or reducing at least one adverse or negative effect orsymptom of a condition, disease or disorder.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein, and refer to an animal, for example a human, towhom treatment, including prophylactic treatment, with thepharmaceutical composition according to the present invention, isprovided. The term “subject” as used herein refers to human andnon-human animals. The terms “non-human animals” and “non-human mammals”are used interchangeably herein and include all vertebrates, e.g.,mammals, such as non-human primates, (particularly higher primates),sheep, dog, rodent, (e.g., mouse or rat), guinea pig, goat, pig, cat,rabbits, cows, horses and non-mammals such as reptiles, amphibians,chickens, and turkeys.

Despite the remarkable activity of CD19-directed chimeric antigenreceptor T cell (CART19) therapy in treating B cell malignancies,limitations include 1) the development of life-threatening complicationssuch as neurotoxicity (NT) and cytokine release syndrome (CRS) and 2)lack of durable response. Emerging literature suggests that inhibitorymyeloid cells and their cytokines play an important role in inducingCART cell toxicities and contribute to CART inhibition. Specifically,and of relevance to the disclosure herein, granulocyte-macrophagecolony-stimulating factor (GM-CSF) was found to be implicated in thedevelopment of NT and CRS after CART19 therapy based on correlativestudies from pivotal clinical trials.

GM-CSF is produced by macrophages, T cells, NK cells, endothelial cellsand fibroblasts and plays several roles in the hematopoietic and immunesystem. GM-CSF plays a redundant role in stimulating stem cells todifferentiate into monocytes, granulocytes, and neutrophils. GM-CSF alsoactivates monocytes and differentiates them into macrophages and is acomponent of the immune response to infections. Following allogeneictransplantation, GM-CSF has also been demonstrated to drive graft versushost pathology by licensing donor derived myeloid cells to produceinflammatory mediators such as interleukin 1β. GM-CSF was also shown torecruit dendritic cells and promote graft versus host disease,amplifying the activation of alloreactive T cells.

In a recent analysis of the pivotal clinical trial ZUMA-1, which led toFDA approval of axicabtagene ciloleucel (Axi-cel; Yescarta®) CART celltherapy, GM-CSF was the most significant cytokine associated with thedevelopment of CRS and NT. GM-CSF was found to be elevated early (withinthe first 24-48 hours) following CART19 infusion, suggesting a potentialrole in initiation and/or propagation of CART cell associatedtoxicities. Preclinical studies identified that depletion of GM-CSFprevents CRS and NT and enhances CART cell anti-tumor activity inpreclinical models. Specifically, these models indicate that GM-CSFneutralization with lenzilumab reduces monocyte activation and decreasesinhibitory myeloid cytokines, which in turn ameliorates the developmentof CRS, preserves blood brain barrier integrity, and preventsneuroinflammation. This preclinical work led to the launch of ZUMA-19,an ongoing phase Ib/II multi-center study of Axi-cel CART19 in asequenced therapy with lenzilumab for GM-CSF neutralization in patientswith relapsed or refractory large B-cell lymphoma (NCT04314843).

While GM-CSF is secreted primarily by myeloid cells and contributes totheir activation, it is also produced by T cells. Therefore, it washypothesized that disruption of GM-CSF during CART cell manufacturingwould result in reduced GM-CSF levels and decreased monocyte activation.Indeed, it was shown that CRISPR/Cas9 gene disruption of GM-CSF in CARTcells during their manufacturing generates GM-CSF^(k/o) CART19 cells,which produce less GM-CSF while maintaining their effector functions.This work was recently corroborated by a preclinical study using TALENsto knockout GM-CSF. In fact, the results indicate that GM-CSF^(k/o)CART19 cells exhibit superior antitumor activity in xenograft modelswhere myeloid cells are lacking. This pointed to a direct impact ofGM-CSF on T cells, independent of its role as a mediator of monocyteactivation and monocyte induced T cell inhibition. To this end, GM-CSFproducing T cells (Th^(GM)) have been identified as a novel subset of Tcells with unique phenotypic and functional properties. Specifically,Th^(GM) cells were found to induce activation of other T cell subsets,thus amplifying T cell responses. Th^(GM) were also demonstrated to bemore susceptible to apoptosis and activation-induced cell death (AICD).

It was, therefore, hypothesized that GM-CSF depletion in CART cellsresults in reduced AICD and enhanced anti-tumor activity independent ofthe effect on myeloid cell activation. In Example 6, how GM-CSFdisruption of CART cells impact their functions directly was tested.

This document provides methods and materials for generating T cells(e.g., chimeric antigen receptor (CAR) T cells (CARTs)) having a reducedexpression level of one or more cytokine polypeptides (e.g., GM-CSFpolypeptides). In some cases, a T cell (e.g., CART) can be engineered toknock out (KO) a nucleic acid encoding a GM-CSF polypeptide to reduceGM-CSF polypeptide expression in that T cell (e.g., as compared to a Tcell that is not engineered to KO a nucleic acid encoding a GM-CSFpolypeptide). A T cell that is engineered to KO a nucleic acid encodinga GM-CSF polypeptide can also be referred to herein as a GM-CSF KO Tcell. In some cases, the methods and materials provided herein can beused to modulate myeloid cells. In some cases, the methods and materialsprovided herein can be used to deplete myeloid cells. In some cases, themethods and materials provided herein can be used to enhance T cell(e.g., CARTs) efficacy.

T cells (e.g., CARTs) provided herein can be designed to have a reducedexpression level of any appropriate cytokine polypeptide or combinationof cytokine polypeptides. For example, a T cell (e.g., a CART) providedherein can be designed to have a reduced expression level of a GM-CSFpolypeptide, an interleukin 6 (IL-6) polypeptide, a G-CSF, a interferongamma (IFN-g) polypeptide, an IL-1B polypeptide, an IL-10 polypeptide, amonocyte chemoattractant protein 1 (MCP-1) polypeptide, a monokineinduced by gamma (MIG) polypeptide, a macrophage inflammatory protein(MIP) polypeptide (e.g., a MIP-1β polypeptide), a tumor necrosis factoralpha (TNF-α) polypeptide, an IL-2 polypeptide, a perforin polypeptide,or any combination thereof. For example, a T cell can be designed tohave a reduced expression level of both GM-CSF and IL-6 polypeptides.

In one aspect, this invention provides a method for enhancing anti-tumorefficacy of immunotherapy in a subject, the method comprisingadministering to the subject CAR-T cells having a GM-CSF geneinactivation, GM-CSF gene knock-down or gene knockout (GM-CSF^(k/o)CAR-T cells), wherein administration of the CAR-T cells improvesanti-tumor efficacy and reduces or prevents immunotherapy-relatedtoxicity. In an embodiment of the herein provided method, the methodfurther comprises administering to the subject an anti-hGM-CSF antibody,wherein the anti-hGM-CSF antibody is a recombinant anti-hGM-CSF antibodythat binds to and neutralizes human GM-CSF. In a particular embodiment,the method comprises administering to the subject an anti-hGM-CSFantibody. In another embodiment, the immunotherapy-related toxicityCAR-T comprises Cytokine Release Syndrome (CRS), neurotoxicity (NT),neuroinflammation or a combination thereof.

In certain embodiments of the herein provided methods, theadministration of (i) the CAR-T cells having a GM-CSF gene inactivation,GM-CSF gene knock-down or gene knockout (GM-CSF ° CAR-T cells) or (ii)the CAR-T cells and the anti-hGM-CSF antibody decreases or preventsCD14+ myeloid cell trafficking to a central nervous system (CNS) of thesubject. In an embodiment, a high level of CD14+ myeloid cells in thecentral nervous system (CNS) of the subject is indicative ofneurotoxicity. A level of CD14+ myeloid cells in the CNS is determinedby performing a lumbar puncture, removing a sample of cerebrospinalfluid (CSF), and measuring the CD14+ cells in the CSF, for example bycytometric flow analysis (Flow Cytometry), ELISA, anti-CD14-FITCmonoclonal antibody or other suitable measurement techniques.

In an embodiment of the herein provided methods, an objective responserate of the subject administered the anti-hGM-CSF antibody is improvedcompared to a subject that is not administered the anti-hGM-CSFantibody. In a specific embodiment, the objective response rate is acomplete response rate or a partial response rate. In anotherembodiment, a progression free response and/or survival of the subjectis improved compared to a subject that is not administered theanti-hGM-CSF antibody and/or the CAR-T cells having a GM-CSF geneinactivation, GM-CSF gene knock-down or gene knockout (GM-CSF^(k/o)CAR-T cells). In a further embodiment, the survival is overall survivalof the subject. In another embodiment, the anti-hGM-CSF antibody isadministered to the subject before, during or after administration ofthe CAR-T cells having a GM-CSF gene inactivation, GM-CSF geneknock-down or GM-CSF^(k/o) CAR-T cells.

In a particular embodiment of the herein provided methods, theimmunotherapy comprises administering chimeric antigenreceptor-expressing T-cells (CAR T-cells). In an embodiment, wherein theCAR T-cells are CART19 cells. In another embodiment of the hereinprovided methods, of claim 1, the immunotherapy comprises adoptive celltransfer selected from the group consisting of administering T-cellreceptor (TCR) modified T-cells, tumor-infiltrating lymphocytes (TIL),chimeric antigen receptor (CAR)-modified natural killer cells, ordendritic cells, or any combination thereof. In some embodiments, theimmunotherapy comprises administration of a monoclonal antibody, acytokine, a cancer vaccine, a T cell engaging bispecific antibody, orany combination thereof. In a particular embodiment, the subject has acancer. In certain embodiments, the cancer is lymphoma or a leukemia. Inanother embodiment, the lymphoma is a diffuse large B cell lymphoma(DLBCL). In still another embodiment, the leukemia is acutelymphoblastic leukemia (ALL). In still another embodiment, the lymphomais mantle cell lymphoma. In still another embodiment, the lymphoma isfollicular lymphoma. In still another embodiment, the cancer is multiplemyeloma.

In another aspect, this invention provides a method for reducing a levelof a non-GM-CSF cytokine in a subject treated with immunotherapy, themethod comprising administering to the subject CAR-T cells having aGM-CSF gene inactivation, GM-CSF gene knock-down or gene knockout(GM-CSF^(k/o) CAR-T cells). In a specific embodiment of the hereinprovided method, the method further comprising administering to thesubject an anti-hGM-CSF antibody to the subject. In another embodiment,the non-GM-CSF cytokine is IP-10, IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5,IL-6, IL-1Ra, IL-9, IL-10, VEGF, TNF-α, FGF-2, IFN-γ, IL-12p40,IL-12p70, sCD40L, KC, MDC, MCP-1, MIP-1a, MIP-1b or a combinationthereof. In another embodiment of the herein provided methods, theimmunotherapy-related toxicity CAR-T comprises CRS, NT,neuroinflammation or a combination thereof.

In one aspect, this invention provides a method for treating orpreventing CAR-T cell related toxicity in a subject in need thereof, themethod comprising administering to the subject CAR-T cells having aGM-CSF gene inactivation, GM-CSF gene knock-down or gene knockout(GM-CSF^(k/o) CAR-T cells). In some embodiments of the herein providedmethods, the CAR-T cell related toxicity comprises neurotoxicity,cytokine release syndrome (CRS) or a combination thereof. In particularembodiments, the subject has a cancer and/or a tumor. In an embodiment,the cancer is lymphoma or a leukemia. In certain embodiments, thelymphoma is a diffuse large B cell lymphoma (DLBCL). In someembodiments, the leukemia is acute lymphoblastic leukemia (ALL). Instill another embodiment, the lymphoma is mantle cell lymphoma. In stillanother embodiment, the lymphoma is follicular lymphoma. In stillanother embodiment, the cancer is multiple myeloma.

In an embodiment of the herein provided methods, levels of the CAR-Tcells having a GM-CSF gene inactivation, GM-CSF gene knock-down or geneknockout (GM-CSF^(k/o) CAR-T cells) expand and persist in blood of thesubject from a peak level of GM-CSF^(k/o) CAR-T cell expansion duringthe first 30 days after administration of the GM-CSF^(k/o) CAR-T cellsand expansion of the GM-CSF^(k/o) CAR-T cells up to at least 90 days to180 days after the administration of the GM-CSF^(k/o) CAR-T cells. Insome embodiments, GM-CSF^(k/o) CAR-T cell expansion and persistence inthe blood of the subject continues for up to 24 months afteradministration of the GM-CSF^(k/o) CAR-T cells. In certain embodiments,GM-CSF^(k/o) CAR-T cell expansion and persistence in the blood of thesubject achieves an anti-cancer or anti-tumor efficacy from 90 days to24 months after administration of the GM-CSF^(k/o) CAR-T cells. In aparticular embodiment, the anti-cancer or anti-tumor efficacy in thesubject is a complete or partial remission of the cancer and/or thetumor. In further particular embodiments, the anti-cancer or anti-tumorefficacy in the subject is a reduction or an absence of signs andsymptoms of the cancer and/or the tumor.

In another aspect, this invention provides a method for increasing CAR-Tcell proliferation in a subject treated with GM-CSF-inactivated orGM-CSF^(k/o) CAR-T cells, the method comprising administering to thesubject CAR-T cells having a GM-CSF gene inactivation, GM-CSF geneknock-down or gene knockout (GM-CSF^(k/o) CAR-T cells), whereinadministration of the GM-CSF^(k/o) CAR-T cells increases CAR-Tproliferation in the subject. In some embodiments, of the hereinprovided methods, the administration of the GM-CSF^(k/o) CAR-T cells andexpansion of the GM-CSF^(k/o) CAR-T cells reduces the overall productionof GM-CSF by CAR T cells by 75%-95%. In an embodiment of the hereinprovided methods, the administration of the GM-CSF^(k/o) CAR-T cells andexpansion of the GM-CSF^(k/o) CAR-T cells reduces the overall productionof GM-CSF by CAR T cells by 95%-99% or eliminates production of GM-CSFby the GM-CSF^(k/o) CAR-T cells. In some embodiments, production ofGM-CSF by the administered GM-CSF^(k/o) CAR T cells is completelyeliminated. In some embodiments of the herein provided methods,reduction or elimination of the production of GM-CSF by the GM-CSF^(k/o)CAR-T cells increases production and expansion of the GM-CSF by theGM-CSF^(k/o) CAR-T cells. In certain embodiments, increased productionand expansion of the GM-CSF by the GM-CSF^(k/o) CAR-T cells reduces ofeliminates CAR-T cell related toxicity in the subject, wherein the CAR-Tcell related toxicity comprises neurotoxicity, cytokine release syndrome(CRS) or a combination thereof. In a particular embodiment, the subjecthas a cancer and/or a tumor. In some embodiments, the cancer is lymphomaor a leukemia. In certain embodiments, the lymphoma is a diffuse large Bcell lymphoma (DLBCL). In some embodiments, the leukemia is acutelymphoblastic leukemia (ALL). In still another embodiment, the lymphomais mantle cell lymphoma. In still another embodiment, the lymphoma isfollicular lymphoma. In still another embodiment, the cancer is multiplemyeloma. In an embodiment of the herein provided methods, levels of theCAR-T cells having a GM-CSF gene inactivation, GM-CSF gene knock-down orgene knockout (GM-CSF^(k/o) CAR-T cells) expand and persist in blood ofthe subject from a peak level of GM-CSF^(k/o) CAR-T cell expansionduring the first 30 days after administration of the GM-CSF^(k/o) CAR-Tcells and expansion of the GM-CSF^(k/o) CAR-T cells up to at least 90days to 180 days after the administration of the GM-CSF^(k/o) CAR-Tcells. In particular embodiments, GM-CSF^(k/o) CAR-T cell expansion andpersistence in the blood of the subject continues for up to 24 monthsafter administration of the GM-CSF^(k/o) CAR-T cells. In certainembodiments, GM-CSF^(k/o) CAR-T cell expansion and persistence in theblood of the subject achieves an anti-cancer or anti-tumor efficacy from90 days to 24 months after administration of the GM-CSF^(k/o) CAR-Tcells. In some embodiments, the anti-cancer or anti-tumor efficacy inthe subject is a complete or partial remission of the cancer and/or thetumor. In a particular embodiment, the anti-cancer or anti-tumorefficacy in the subject is a reduction or an absence of signs andsymptoms of the cancer and/or the tumor.

In one aspect, this invention provides a method for GM-CSF geneinactivation, GM-CSF gene knock-down or GM-CSF knockout (KO) in a cellcomprising targeted genome editing or GM-CSF gene silencing. In anembodiment of the herein provided method, the method further comprisesan endonuclease as a nucleic acid cutting enzyme. In some embodiments,the endonuclease is a Fok1 restriction enzyme or a flap endonuclease 1(FEN-1). In certain embodiments, the endonuclease is a Cas9 CRISPRassociated protein 9 (Cas9). In a specific embodiment, the GM-CSF geneinactivation by CRISPR/Cas9 targets and edits a GM-CSF gene at Exon 1,Exon 2, Exon 3 or Exon 4. In an embodiment, the GM-CSF gene inactivationcomprising CRISPR/Cas9 targets and edits the GM-CSF gene at Exon 3. Inanother embodiment, the GM-CSF gene inactivation comprising CRISPR/Cas9targets and edits the GM-CSF gene at Exon 1.

In still another embodiment, the GM-CSF gene inactivation comprisesmultiple CRISPR/Cas9 enzymes, wherein each Cas9 enzyme targets and editsa different sequence of the GM-CSF gene at Exon 1, Exon 2, Exon 3 orExon 4. In another embodiment, the GM-CSF gene inactivation comprisesbi-allelic CRISPR/Cas9 targeting and knockout/inactivation of the GM-CSFgenes.

In a particular embodiment of the herein provided methods, the methodfurther comprises treating primary T cells with valproic acid to enhancebi-allele gene knockout/inactivation. In another embodiment the targetedgenome editing comprises Zinc finger (ZnF) proteins. In certainembodiments, the targeted genome editing comprises transcriptionactivator-like effector nucleases (TALENS). In particular embodiments,the targeted genome editing comprises a homing endonuclease, wherein thehoming endonuclease is an ARC nuclease (ARCUS) or a meganuclease. In anembodiment of the herein provided methods, the targeted genome editingcomprises a flap endonuclease (FEN-1). In a specific embodiment, thecell is a CAR T cell. In a particular embodiment, the CAR T cell is aCD19 CAR-T cell. In another embodiment, the CAR T cell is a BCMA CAR-Tcell. In another embodiment, the GM-CSF gene silencing is selected fromthe group consisting of RNA interference (RNAi), short interfering RNS(siRNA), and DNA-directed RNA interference (ddRNAi).

In one aspect, this invention provides a method for making a chimericantigen receptor T cell having a reduced level of granulocyte-macrophagecolony-stimulating factor (GM-CSF) polypeptides, said method comprising:introducing a nucleic acid construct into an ex vivo T cell, whereinsaid nucleic acid construct comprises: a) a nucleic acid encoding aguide RNA, wherein said guide RNA is complementary to a GM-CSF messengerRNA; b) a nucleic acid encoding a Cas nuclease, and c) a nucleic acidencoding said chimeric antigen receptor. In an embodiment of said hereinprovided method, said guide RNA comprises a nucleic acid sequence setforth in SEQ ID NO:1. In another embodiment, said Cas nuclease is Cas9nuclease. In a further embodiment, said nucleic acid encoding saidchimeric antigen receptor comprises a nucleic acid sequence set forth inSEQ ID NO:2. In another embodiment, said nucleic acid construct is aviral vector. In a particular embodiment, said viral vector is alentiviral vector. In a further embodiment, said chimeric antigenreceptor targets a tumor-associated antigen. In another embodiment, saidtumor-associated antigen is CD19. In a further embodiment, saidintroducing step comprises transduction.

In another aspect, this invention provides a method for making achimeric antigen receptor T cell having a reduced level ofgranulocyte-macrophage colony-stimulating factor (GM-CSF) polypeptides,said method comprising: introducing a complex into an ex vivo T cell,wherein said complex comprises: a) a guide RNA, wherein said guide RNAis complementary to a GM-CSF messenger RNA; and b) a Cas nuclease; andintroducing a nucleic acid encoding said chimeric antigen receptor intosaid ex vivo T cell. In an embodiment of the herein provided method,said guide RNA comprises a nucleic acid sequence set forth in SEQ IDNO:1. In another embodiment, said Cas nuclease is Cas9 nuclease. In aparticular embodiment, said nucleic acid encoding said chimeric antigenreceptor comprises a nucleic acid sequence set forth in SEQ ID NO:2. Inanother embodiment, said complex is a ribonucleoprotein. In a furtherembodiment, said chimeric antigen receptor targets a tumor-associatedantigen. In a particular embodiment, said tumor-associated antigen isCD19. In another embodiment of the herein provided method, saidintroducing steps comprise electroporation.

In another aspect, this invention provides a method for treating amammal having cancer, wherein said method comprises administeringchimeric antigen receptor T cells having a reduced level ofgranulocyte-macrophage colony-stimulating factor (GM-CSF) polypeptidesto said mammal. In a particular embodiment, said mammal is a human. Inanother embodiment, said cancer is a lymphoma. In a further embodiment,said lymphoma is a diffuse large B cell lymphoma.

In another embodiment, said cancer is a leukemia. In another embodiment,said leukemia is an acute lymphoblastic leukemia. In still anotherembodiment, the lymphoma is mantle cell lymphoma. In still anotherembodiment, the lymphoma is follicular lymphoma. In still anotherembodiment, the cancer is multiple myeloma. In an embodiment, saidchimeric antigen receptor targets a tumor-associated antigen. In aparticular embodiment, said tumor-associated antigen is CD19. In aparticular embodiment, the tumor-associated antigen is BMCA.

In an aspect, this invention provides a method for making a chimericantigen receptor T cell having a reduced level of cytokine polypeptides,said method comprising: introducing a nucleic acid construct into an exvivo T cell, wherein said nucleic acid construct comprises: a) a nucleicacid encoding a guide RNA, wherein said guide RNA is complementary to acytokine messenger RNA; b) a nucleic acid encoding a Cas nuclease, andc) a nucleic acid encoding said chimeric antigen receptor. In certainembodiments of said herein provided method, said cytokine polypeptidescomprise granulocyte-macrophage colony-stimulating factor (GM-CSF)polypeptides and/or interleukin 6 (IL-6) polypeptides. In a particularembodiment, said cytokine polypeptides are GM-CSF polypeptides, andwherein said guide RNA comprises a nucleic acid sequence set forth inSEQ ID NO:1. In an embodiment, said Cas nuclease is Cas9 nuclease. Inanother embodiment, said nucleic acid encoding said chimeric antigenreceptor comprises a nucleic acid sequence set forth in SEQ ID NO:2. Ina further embodiment, said nucleic acid construct is a viral vector. Ina specific embodiment, said viral vector is a lentiviral vector. Inanother embodiment, said chimeric antigen receptor targets atumor-associated antigen. In various embodiments, said tumor-associatedantigen is CD19. In another embodiment, said introducing step comprisestransduction.

In another aspect, this invention provides a method for making achimeric antigen receptor T cell having a reduced level of cytokinepolypeptides, said method comprising: introducing a complex into an exvivo T cell, wherein said complex comprises: a) a guide RNA, whereinsaid guide RNA is complementary to a cytokine messenger RNA; and b) aCas nuclease; and introducing a nucleic acid encoding said chimericantigen receptor into said ex vivo T cell. In an embodiment of saidherein provided method, said cytokine polypeptides comprisegranulocyte-macrophage colony-stimulating factor (GM-CSF) polypeptidesand/or interleukin 6 (IL-6) polypeptides. In a particular embodiment,said cytokine polypeptides are GM-CSF polypeptides, and wherein saidguide RNA comprises a nucleic acid sequence set forth in SEQ ID NO:1. Inanother embodiment, said Cas nuclease is Cas9 nuclease. In anotherembodiment, said nucleic acid encoding said chimeric antigen receptorcomprises a nucleic acid sequence set forth in SEQ ID NO:2. In a furtherembodiment, said complex is a ribonucleoprotein. In a still furtherembodiment, said chimeric antigen receptor targets a tumor-associatedantigen. In a specific embodiment, said tumor-associated antigen isCD19. In another embodiment, said introducing steps compriseselectroporation.

In still another aspect, this invention provides a method for treating amammal having cancer, wherein said method comprises administeringchimeric antigen receptor T cells having a reduced level of cytokinepolypeptides to said mammal. In an embodiment of said provided method,said cytokine polypeptides comprise granulocyte-macrophagecolony-stimulating factor (GM-CSF) polypeptides and/or interleukin 6(IL-6) polypeptides. In another embodiment, said cytokine polypeptidesare GM-CSF polypeptides, and wherein said guide RNA comprises a nucleicacid sequence set forth in SEQ ID NO:1. In a further embodiment, saidmammal is a human. In another embodiment, said cancer is a lymphoma. Instill another embodiment, said lymphoma is a diffuse large B celllymphoma. In another embodiment, said cancer is a leukemia. In a furtherembodiment, said leukemia is an acute lymphoblastic leukemia. In stillanother embodiment, the lymphoma is mantle cell lymphoma. In stillanother embodiment, the lymphoma is follicular lymphoma. In stillanother embodiment, the cancer is multiple myeloma. In an embodiment,said chimeric antigen receptor targets a tumor-associated antigen. In aparticular embodiment, said tumor-associated antigen is CD19. In aparticular embodiment, said tumor-associated antigen is BCMA.

In one aspect, this invention provides a method for improving T celleffector functions of a chimeric antigen receptor T cell, said methodcomprising: introducing a nucleic acid construct into an ex vivo T cell,wherein said nucleic acid construct comprises: a) a nucleic acidencoding a guide RNA, wherein said guide RNA is complementary to aGM-CSF messenger RNA; b) a nucleic acid encoding a Cas nuclease, and c)a nucleic acid encoding said chimeric antigen receptor.

In another aspect, this invention provides a method for improving T celleffector functions of a chimeric antigen receptor T cell, said methodcomprising: introducing a complex into an ex vivo T cell, wherein saidcomplex comprises: a) a guide RNA, wherein said guide RNA iscomplementary to a GM-CSF messenger RNA; and b) a Cas nuclease; andintroducing a nucleic acid encoding said chimeric antigen receptor intosaid ex vivo T cell.

The term “reduced level” as used herein with respect to an expressionlevel of a cytokine (e.g., GM-CSF) refers to any level that is lowerthan a reference expression level of that cytokine (e.g., GM-CSF). Theterm “reference level” as used herein with respect to a cytokine (e.g.,GM-CSF) refers to the level of that cytokine (e.g., GM-CSF) typicallyobserved in a sample (e.g., a control sample) from one or more mammals(e.g., humans) not engineered to have a reduced expression level of thatcytokine (e.g., GM-CSF polypeptides) as described herein. Controlsamples can include, without limitation, T cells that are wild-type Tcells (e.g., T cells that are not GM-SCF KO T cells). In some cases, areduced expression level of a cytokine polypeptide (e.g., a GM-CSFpolypeptide) can be an undetectable level of that cytokine (e.g.,GM-CSF). In some cases, a reduced expression level of GM-CSFpolypeptides can be an eliminated level of GM-CSF.

In some cases, a T cell having (e.g., engineered to have) a reducedexpression level of one or more cytokine polypeptides such as a GM-CSFKO T cell can maintain normal T cell functions such as T celldegranulation and release of cytokines (e.g., as compared to a CART thatis not engineered to have a reduced expression level of that cytokine(e.g., GM-CSF polypeptides) as described herein).

In some cases, a T cell having (e.g., engineered to have) a reducedlevel of GM-CSF polypeptides (e.g., a GM-CSF KO T cell) can haveenhanced CART function such as antitumor activity, proliferation, cellkilling, cytokine production, exhaustion susceptibility, antigenspecific effector functions, persistence, and differentiation (e.g., ascompared to a CART that is not engineered to have a reduced level ofGM-CSF polypeptides as described herein).

In some cases, a T cell having (e.g., engineered to have) a reducedlevel of GM-CSF polypeptides (e.g., a GM-CSF KO T cell) can haveenhanced T cell expansion (e.g., as compared to a CART that is notengineered to have a reduced level of GM-CSF polypeptides as describedherein).

A T cell having (e.g., engineered to have) a reduced expression level ofone or more cytokines (e.g., a GM-CSF polypeptide) such as a GM-CSF KO Tcell can be any appropriate T cell. A T cell can be a naive T cell.Examples of T cells that can be designed to have a reduced expressionlevel of one or more cytokines as described herein include, withoutlimitation, cytotoxic T cells (e.g., CD4+ CTLs and/or CD8+ CTLs). Forexample, a T cell that can be engineered to have a reduced level ofGM-CSF polypeptides as described herein can be a CART. In some cases,one or more T cells can be obtained from a mammal (e.g., a mammal havingcancer). For example, T cells can be obtained from a mammal to betreated with the materials and method described herein.

A T cell having (e.g., engineered to have) a reduced expression level ofone or more cytokine polypeptides (e.g., a GM-CSF polypeptide) such as aGM-CSF KO T cell can be generated using any appropriate method. In somecases, a T cell (e.g., CART) can be engineered to KO a nucleic acidencoding a GM-CSF polypeptide to reduce GM-CSF polypeptide expression inthat T cell.

In some cases, when a T cell (e.g., CART) is engineered to KO a nucleicacid encoding a cytokine (e.g., a GM-CSF polypeptide) to reduceexpression of that cytokine polypeptide in that T cell, any appropriatemethod can be used to KO a nucleic acid encoding that cytokine. Examplesof techniques that can be used to knock out a nucleic acid sequenceencoding a cytokine polypeptide (e.g., a GM-CSF polypeptide) include,without limitation, gene editing, homologous recombination,non-homologous end joining, and microhomology end joining. For example,gene editing (e.g., with engineered nucleases) can be used to KO anucleic acid encoding a GM-CSF polypeptide. Nucleases useful for genomeediting include, without limitation, CRISPR-associated (Cas) nucleases,zinc finger nucleases (ZFNs), transcription activator-like effector(TALE) nucleases, and homing endonucleases (HE; also referred to asmeganucleases).

In some cases, a clustered regularly interspaced short palindromicrepeat (CRISPR)/Cas system can be used (e.g., can be introduced into oneor more T cells) to KO a nucleic acid encoding cytokine polypeptide(e.g., a GM-CSF polypeptide) (see, e.g., FIG. 1 and Example 1). ACRISPR/Cas system used to KO a nucleic acid encoding a cytokinepolypeptide (e.g., a GM-CSF polypeptide) can include any appropriateguide RNA (gRNA). In some cases, a gRNA can be complementary to anucleic acid encoding a GM-CSF polypeptide (e.g., a GM-CSF mRNA).Examples of gRNAs that are specific to a nucleic acid encoding a GM-CSFpolypeptide include, without limitation, GACCTGCCTACAGACCCGCC (SEQ IDNO:1), GCAGTGCTGCTTGTAGTGGC (SEQ ID NO:10), TCAGGAGACGCCGGGCCTCC (SEQ IDNO:3), CAGCAGCAGTGTCTCTACTC (SEQ ID NO:4), CTCAGAAATGTTTGACCTCC (SEQ IDNO:5), and GGCCGGTCTCACTCCTGGAC (SEQ ID NO:6). In some cases, a gRNAcomponent of a CRISPR/Cas system designed to KO a nucleic acid encodinga GM-CSF polypeptide can include the nucleic acid sequence set forth inSEQ ID NO:1.

A CRISPR/Cas system used to KO a nucleic acid encoding a cytokinepolypeptide (e.g., a GM-CSF polypeptide) can include any appropriate Casnuclease. Examples of Cas nucleases include, without limitation, Cas1,Cas2, Cas3, Cas9, Cas10, and Cpf1. In some cases, a Cas component of aCRISPR/Cas system designed to KO a nucleic acid encoding a cytokinepolypeptide (e.g., a GM-CSF polypeptide) can be a Cas9 nuclease. Forexample, the Cas9 nuclease of a CRISPR/Cas9 system described herein canbe a lentiCRISPRv2 (see, e.g., Shalem et al., 2014 Science 343:84-87;and Sanjana et al., 2014 Nature methods 11: 783-784, each of which isincorporated herein by reference in its entirety).

Components of a CRISPR/Cas system (e.g., a gRNA and a Cas nuclease) usedto KO a nucleic acid encoding a cytokine polypeptide (e.g., a GM-CSFpolypeptide) can be introduced into one or more T cells (e.g., CARTs) inany appropriate format. In some cases, a component of a CRISPR/Cassystem can be introduced into one or more T cells as a nucleic acidencoding a gRNA and/or a nucleic acid encoding a Cas nuclease. Forexample, a nucleic acid encoding at least one gRNA (e.g., a gRNAsequence specific to a nucleic acid encoding a GM-CSF polypeptide) and anucleic acid at least one Cas nuclease (e.g., a Cas9 nuclease) can beintroduced into one or more T cells. In some cases, a component of aCRISPR/Cas system can be introduced into one or more T cells as a gRNAand/or as a Cas nuclease. For example, at least one gRNA (e.g., a gRNAsequence specific to a nucleic acid encoding a GM-CSF polypeptide) andat least one Cas nuclease (e.g., a Cas9 nuclease) can be introduced intoone or more T cells.

In some cases, when components of a CRISPR/Cas system (e.g., a gRNA anda Cas nuclease) are introduced into one or more T cells as nucleic acidencoding the components (e.g., nucleic acid encoding a gRNA and nucleicacid encoding a Cas nuclease), the nucleic acid can be any appropriateform. For example, a nucleic acid can be a construct (e.g., anexpression construct). A nucleic acid encoding at least one gRNA and anucleic acid encoding at least one Cas nuclease can be on separatenucleic acid constructs or on the same nucleic acid construct. In somecases, a nucleic acid encoding at least one gRNA and a nucleic acidencoding at least one Cas nuclease can be on a single nucleic acidconstruct. A nucleic acid construct can be any appropriate type ofnucleic acid construct. Examples of nucleic acid constructs that can beused to express at least one gRNA and/or at least one Cas nucleaseinclude, without limitation, expression plasmids and viral vectors(e.g., lentiviral vectors). In cases where a nucleic acid encoding atleast one gRNA and a nucleic acid encoding at least one Cas nuclease areon separate nucleic acid constructs, the nucleic acid constructs can bethe same type of construct or different types of constructs. In somecases, a nucleic acid encoding at least one gRNA sequence specific to anucleic acid encoding a cytokine polypeptide (e.g., a GM-CSFpolypeptide) and a nucleic acid encoding at least one Cas nuclease canbe on a single lentiviral vector. For example, a lentiviral vectorencoding at least one gRNA sequence specific to a nucleic acid encodinga cytokine polypeptide (e.g., GM-CSF polypeptide), encoding at least onegRNA including the sequence set forth in SEQ ID NO:1, and encoding atleast one Cas9 nuclease can be used in ex vivo engineering of T cells tohave a reduced expression level of that cytokine (e.g., a GM-CSFpolypeptide).

In some cases, components of a CRISPR/Cas system (e.g., a gRNA and a Casnuclease) can be introduced directly into one or more T cells (e.g., asa gRNA and/or as Cas nuclease). A gRNA and a Cas nuclease can beintroduced into the one or more T cells separately or together. In caseswhere a gRNA and a Cas nuclease are introduced into the one or more Tcells together, the gRNA and the Cas nuclease can be in a complex. Whena gRNA and a Cas nuclease are in a complex, the gRNA and the Casnuclease can be covalently or non-covalently attached. In some cases, acomplex including a gRNA and a Cas nuclease also can include one or moreadditional components. Examples of complexes that can include componentsof a CRISPR/Cas system (e.g., a gRNA and a Cas nuclease) include,without limitation, ribonucleoproteins (RNPs) and effector complexes(e.g., containing a CRISPR RNAs (crRNAs) a Cas nuclease). For example,at least one gRNA and at least one Cas nuclease can be included in aRNP. In some cases, a RNP can include gRNAs and Cas nucleases at a ratioof about 1:1 to about 10:1 (e.g., about 1:1 to about 10:1, about 2:1 toabout 10:1, about 3:1 to about 10:1, about 5:1 to about 10:1, about 8:1to about 10:1, about 1:1 to about 9:1, about 1:1 to about 7:1, about 1:1to about 5:1, about 1:1 to about 4:1, about 1:1 to about 3:1, about 1:1to about 2:1, about 2:1 to about 8:1, about 3:1 to about 6:1, about 4:1to about 5:1, or about 5:1 to about 7:1). For example, a RNP can includegRNAs and Cas nucleases at about a 1:1 ratio. For example, a RNP caninclude gRNAs and Cas nucleases at about a 2:1 ratio. In some cases, aRNP including at least one gRNA sequence specific to a nucleic acidencoding a GM-CSF polypeptide (e.g., encoding at least one gRNAincluding the sequence set forth in SEQ ID NO:1) and at least one Cas9nuclease can be used in ex vivo engineering of T cells to have a reducedlevel of GM-CSF polypeptides.

Components of a CRISPR/Cas system (e.g., a gRNA and a Cas nuclease) usedto KO a nucleic acid encoding a cytokine polypeptide (e.g., a GM-CSFpolypeptide) can be introduced into one or more T cells (e.g., CARTs)using any appropriate method. A method of introducing components of aCRISPR/Cas system into a T cell can be a physical method. A method ofintroducing components of a CRISPR/Cas system into a T cell can be achemical method. A method of introducing components of a CRISPR/Cassystem into a T cell can be a particle-based method. Examples of methodsthat can be used to introduce components of a CRISPR/Cas system into oneor more T cells include, without limitation, electroporation,transfection (e.g., lipofection), transduction (e.g., viral vectormediated transduction), microinjection, and nucleofection. In somecases, when components of a CRISPR/Cas system are introduced into one ormore T cells as nucleic acid encoding the components, the nucleic acidencoding the components can be transduced into the one or more T cells.For example, a lentiviral vector encoding at least one gRNA sequencespecific to a nucleic acid encoding a GM-CSF polypeptide (e.g., encodingat least one gRNA including the sequence set forth in SEQ ID NO:1) andat least one Cas9 nuclease can be transduced into T cells (e.g., ex vivoT cells). In some cases, when components of a CRISPR/Cas system areintroduced directly into one or more T cells, the components can beelectroporated into the one or more T cells. For example, a RNPincluding at least one gRNA sequence specific to a nucleic acid encodinga GM-CSF polypeptide (e.g., encoding at least one gRNA including thesequence set forth in SEQ ID NO:1) and at least one Cas9 nuclease can beelectroporated into T cells (e.g., ex vivo T cells). In some cases,components of a CRISPR/Cas system can be introduced ex vivo into one ormore T cells. For example, ex vivo engineering of T cells have a reducedlevel of GM-CSF polypeptides can include transducing isolated T cellswith a lentiviral vector encoding components of a CRISPR/Cas system. Forexample, ex vivo engineering of T cells having reduced levels of GM-CSFpolypeptides can include electroporating isolated T cells with a complexincluding components of a CRISPR/Cas system. In cases where T cells areengineered ex vivo to have a reduced level of GM-CSF polypeptides, the Tcells can be obtained from any appropriate source (e.g., a mammal suchas the mammal to be treated or a donor mammal, or a cell line).

In some cases, a T cell (e.g., a CART) can be treated with one or moreinhibitors of GM-CSF polypeptide expression or GM-CSF polypeptideactivity to reduce GM-CSF polypeptide expression in that T cell (e.g.,as compared to a T cell that was not treated with one or more inhibitorsof GM-CSF polypeptide expression or GM-CSF polypeptide activity). Aninhibitor of GM-CSF polypeptide expression or GM-CSF polypeptideactivity can be any appropriate inhibitor. Example of inhibitors ofGM-CSF polypeptide expression or GM-CSF polypeptide activity include,without limitation, nucleic acid molecules designed to induce RNAinterference (e.g., a siRNA molecule or a shRNA molecule), antisensemolecules, miRNAs, receptor blockade, and antibodies (e.g., antagonisticantibodies and neutralizing antibodies).

A T cell having (e.g., engineered to have) a reduced expression level ofone or more cytokines (e.g., a GM-CSF KO T cell) can express (e.g., canbe engineered to express) any appropriate antigen receptor. In somecases, an antigen receptor can be a heterologous antigen receptor. Insome cases, an antigen receptor can be a CAR. In some cases, an antigenreceptor can be a tumor antigen (e.g., tumor-specific antigen) receptor.For example, a T cell can be engineered to express a tumor-specificantigen receptor that targets a tumor-specific antigen (e.g., a cellsurface tumor-specific antigen) expressed by a cancer cell in a mammalhaving cancer. Examples of antigens that can be recognized by an antigenreceptor expressed in a T cell having reduced expression of a cytokinepolypeptide (e.g., a GM-CSF polypeptide) as described herein include,without limitation, cluster of differentiation 19 (CD19), mucin 1(MUC-1), human epidermal growth factor receptor 2 (HER-2), estrogenreceptor (ER), epidermal growth factor receptor (EGFR),alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125,epithelial tumor antigen (ETA), melanoma-associated antigen (MAGE),CD33, CD123, CLL-1, E-Cadherin, folate receptor alpha, folate receptorbeta, IL13R, EGFRviii, CD22, CD20, kappa light chain, lambda lightchain, desmopressin, CD44v, CD45, CD30, CD5, CD7, CD2, CD38, BCMA,CD138, FAP, CS-1, EphA3, EphA2, and C-met. For example, a T cell havinga reduced level of GM-CSF polypeptides can be designed to express anantigen receptor targeting CD19. An exemplary nucleic acid sequenceencoding a CAR targeting CD19 (CAR19) is shown in FIG. 5.

Any appropriate method can be used to express an antigen receptor on a Tcell having (e.g., engineered to have) a reduced expression level of oneor more cytokine polypeptides (e.g., a GM-CSF KO T cell). For example, anucleic acid encoding an antigen receptor can be introduced into one ormore T cells. In some cases, viral transduction can be used to introducea nucleic acid encoding an antigen receptor into a non-dividing a cell.A nucleic acid encoding an antigen receptor can be introduced in a Tcell using any appropriate method. In some cases, a nucleic acidencoding an antigen receptor can be introduced into a T cell bytransduction (e.g., viral transduction using a retroviral vector such asa lentiviral vector) or transfection. In some cases, a nucleic acidencoding an antigen receptor can be introduced ex vivo into one or moreT cells. For example, ex vivo engineering of T cells expressing anantigen receptor can include transducing isolated T cells with alentiviral vector encoding an antigen receptor. In cases where T cellsare engineered ex vivo to express an antigen receptor, the T cells canbe obtained from any appropriate source (e.g., a mammal such as themammal to be treated or a donor mammal, or a cell line).

In some cases, when a T cell having (e.g., engineered to have) a reducedexpression level of one or more cytokine polypeptides (e.g., a GM-CSFgene KO T cell) also expresses (e.g., is engineered to express) anantigen receptor, that T cell can be engineered to have a reducedexpression level of that cytokine and engineered to express an antigenreceptor using any appropriate method. In some cases, a T cell can beengineered to have a reduced expression level of a cytokine polypeptide(e.g., a GM-CSF polypeptide) first and engineered to express an antigenreceptor second, or vice versa. In some cases, a T cell can besimultaneously engineered to have a reduced expression level of one ormore cytokine polypeptides (e.g., a GM-CSF polypeptide) and to expressan antigen receptor. For example, one or more nucleic acids used toreduce expression of a cytokine polypeptide such as a GM-CSF polypeptide(e.g., a lentiviral vector encoding at least one gRNA sequence specificto a nucleic acid encoding that cytokine and at least one Cas9 nucleaseor a nucleic acid encoding at least one oligonucleotide that iscomplementary to that cytokine's mRNA) and one or more nucleic acidsencoding an antigen receptor (e.g., a CAR) can be simultaneouslyintroduced into one or more T cells. One or more nucleic acids used toreduce expression of a cytokine polypeptide (e.g., a GM-CSF polypeptide)and one or more nucleic acids encoding an antigen receptor can beintroduced into one or more T cells on separate nucleic acid constructsor on a single nucleic acid construct. In some cases, one or morenucleic acids used to reduce expression of a cytokine polypeptide (e.g.,a GM-CSF polypeptide) and one or more nucleic acids encoding an antigenreceptor can be introduced into one or more T cells on a single nucleicacid construct. In some cases, one or more nucleic acids used to reduceexpression of a cytokine polypeptide (e.g., a GM-CSF polypeptide) andone or more nucleic acids encoding an antigen receptor can be introducedex vivo into one or more T cells. In cases where T cells are engineeredex vivo to have a reduced expression levels of one or more cytokinepolypeptides (e.g., a GM-CSF polypeptide) and to express an antigenreceptor, the T cells can be obtained from any appropriate source (e.g.,a mammal such as the mammal to be treated or a donor mammal, or a cellline).

In some cases, a T cell having (e.g., engineered to have) a reducedexpression level of one or more cytokine polypeptides (e.g., a GM-CSF KOT cell) can be stimulated. A T cell can be stimulated at the same timeas being engineered to have a reduced level of one or more cytokinepolypeptides or independently of being engineered to have a reducedlevel of one or more cytokine polypeptides. For example, one or more Tcells having a reduced level of GM-CSF polypeptides used in an adoptivecell therapy can be stimulated first, and can be engineered to have areduced expression level of GM-CSF polypeptides second, or vice versa.In some cases, one or more T cells having a reduced expression level ofa cytokine polypeptide (e.g., a GM-CSF polypeptide) used in an adoptivecell therapy can be stimulated first, and can be engineered to have areduced level of that cytokine polypeptide second. A T cell can bestimulated using any appropriate method. For example, a T cell can bestimulated by contacting the T cell with one or more CD polypeptides.Examples of CD polypeptides that can be used to stimulate a T cellinclude, without limitation, CD3, CD28, inducible T cell co-stimulator(ICOS), CD137, CD2, OX40, and CD27. In some cases, a T cell can bestimulated with CD3 and CD28 prior to introducing components of aCRISPR/Cas system (e.g., a gRNA and/or a Cas nuclease) to the T cell toKO a nucleic acid encoding one or more cytokine polypeptides (e.g., aGM-CSF polypeptide).

This document also provides methods and materials involved in treatingcancer. For example, one or more T cells having (e.g., engineered tohave) a reduced expression level of a cytokine polypeptide (e.g., aGM-CSF KO T cells) can be administered (e.g., in an adoptive celltherapy such as a CART therapy) to a mammal (e.g., a human) havingcancer to treat the mammal. In some cases, methods of treating a mammalhaving cancer as described herein can reduce the number of cancer cells(e.g., cancer cells expressing a tumor antigen) within a mammal. In somecases, methods of treating a mammal having cancer as described hereincan reduce the size of one or more tumors (e.g., tumors expressing atumor antigen) within a mammal.

In some cases, administering T cells having (e.g., engineered to have) areduced expression level of a cytokine polypeptide (e.g., a GM-CSF KO Tcell) to a mammal does not result in CRS. For example, administering Tcells having a reduced level of GM-CSF polypeptides to a mammal does notresult in release of cytokines associated with CRS (e.g., CRS criticalcytokines). Examples of cytokines associated with CRS include, withoutlimitation, IL-6, G-CSF, IFN-g, IL-1B, IL-10, MCP-1, MIG, MIP, MIP 1b,TNF-α, IL-2, and perforin.

In some cases, administering T cells having (e.g., engineered to have) areduced expression level of a cytokine polypeptide (e.g., a GM-CSF KO Tcell) to a mammal does not result in neurotoxicity. For example,administering T cells having a reduced level of GM-CSF polypeptides to amammal does not result in differentiation and/or activation of whiteblood cells, the differentiation and/or activation of which, isassociated with neurotoxicity. Examples of white blood cells, thedifferentiation and/or activation of which, is associated withneurotoxicity include, without limitation, monocytes, macrophages,T-cells, dendritic cells, microglia, astrocytes, and neutrophils.

Any appropriate mammal (e.g., a human) having a cancer can be treated asdescribed herein. Examples of mammals that can be treated as describedherein include, without limitation, humans, primates (such as monkeys),dogs, cats, horses, cows, pigs, sheep, mice, and rats. For example, ahuman having a cancer can be treated with one or more T cells having(e.g., engineered to have) a reduced expression level of a cytokinepolypeptide (e.g., a GM-CSF polypeptide) in, for example, an adoptive Tcell therapy such as a CART cell therapy using the methods and materialsdescribed herein.

When treating a mammal (e.g., a human) having a cancer as describedherein, the cancer can be any appropriate cancer. In some cases, acancer treated as described herein can be a solid tumor. In some cases,a cancer treated as described herein can be a hematological cancer. Insome cases, a cancer treated as described herein can be a primarycancer. In some cases, a cancer treated as described herein can be ametastatic cancer. In some cases, a cancer treated as described hereincan be a refractory cancer. In some cases, a cancer treated as describedherein can be a relapsed cancer. In some cases, a cancer treated asdescribed herein can express a tumor-associated antigen (e.g., anantigenic substance produced by a cancer cell). Examples of cancers thatcan be treated as described herein include, without limitation, B cellcancers (e.g., diffuse large B cell lymphoma (DLBCL) and B cellleukemias), acute lymphoblastic leukemia (ALL), chronic lymphocyticleukemia (CLL), follicular lymphoma, mantle cell lymphoma, non-Hodgkinlymphoma, Hodgkin lymphoma, acute myeloid leukemia (AML), multiplemyeloma, head and neck cancers, sarcomas, breast cancer,gastrointestinal malignancies, bladder cancers, urothelial cancers,kidney cancers, lung cancers, prostate cancers, ovarian cancers,cervical cancers, genital cancers (e.g., male genital cancers and femalegenital cancers), and bone cancers. For example, one or more T cellshaving (e.g., engineered to have) a reduced level of GM-CSF polypeptides(e.g., a GM-CSF KO T cells) can be used to treat a mammal having DLBCL.For example, one or more T cells having (e.g., engineered to have) areduced level of GM-CSF polypeptides (e.g., a GM-CSF KO T cells) can beused to treat a mammal having ALL.

Any appropriate method can be used to identify a mammal having cancer.For example, imaging techniques and biopsy techniques can be used toidentify mammals (e.g., humans) having cancer.

Once identified as having a cancer (e.g., DLBCL or ALL), a mammal can beadministered one or more T cells having (e.g., engineered to have) areduced expression level of a cytokine polypeptide (e.g., a GM-CSF KO Tcells) described herein.

For example, one or more T cells having (e.g., engineered to have) areduced expression level of a cytokine polypeptide (e.g., a GM-CSF KO Tcells) can be used in an adoptive T cell therapy (e.g., a CART celltherapy) to treat a mammal having a cancer. For example, one or more Tcells having a reduced level of GM-CSF polypeptides can be used in anadoptive T cell therapy (e.g., a CART cell therapy) targeting anyappropriate antigen within a mammal (e.g., a mammal having cancer). Insome cases, an antigen can be a tumor-associated antigen (e.g., anantigenic substance produced by a cancer cell). Examples oftumor-associated antigens that can be targeted by an adoptive T celltherapy provided herein include, without limitation, CD19 (associatedwith DLBCL, ALL, FL, MCL, and CLL), AFP (associated with germ celltumors and/or hepatocellular carcinoma), CEA (associated with bowelcancer, lung cancer, and/or breast cancer), CA-125 (associated withovarian cancer), MUC-1 (associated with breast cancer), ETA (associatedwith breast cancer), MAGE (associated with malignant melanoma), CD33(associated with AML), CD123 (associated with AML), CLL-1 (associatedwith AML), E-Cadherin (associated with epithelial tumors), folatereceptor alpha (associated with ovarian cancers), folate receptor feta(associated with ovarian cancers and AML), IL13R (associated with braincancers), EGFRviii (associated with brain cancers), CD22 (associatedwith B cell cancers), CD20 (associated with B cell cancers), kappa lightchain (associated with B cell cancers), lambda light chain (associatedwith B cell cancers), CD44v (associated with AML), CD45 (associated withhematological cancers), CD30 (associated with Hodgkin lymphomas and Tcell lymphomas), CD5 (associated with T cell lymphomas), CD7 (associatedwith T cell lymphomas), CD2 (associated with T cell lymphomas), CD38(associated with multiple myelomas and AML), BCMA (associated withmultiple myelomas), CD138 (associated with multiple myelomas and AML),FAP (associated with solid tumors), CS-1 (associated with multiplemyeloma), EphA3 (associated with solid tumors including breast, lung,colon, prostate, renal, glioblastoma multiforme, and melanoma) and c-Met(associated with breast cancer). For example, one or more T cells havinga reduced level of GM-CSF polypeptides can be used in CART cell therapytargeting CD19 (e.g., CART19 cell therapy) to treat cancer as describedherein.

In some cases, one or more T cells having (e.g., engineered to have) areduced expression level of a cytokine polypeptide (e.g., a GM-CSF KO Tcells) can be used in an adoptive T cell therapy (e.g., a CART celltherapy) to treat a mammal having a disease or disorder other thancancer. For example, one or more T cells having a reduced level ofGM-CSF polypeptides can be used in an adoptive T cell therapy (e.g., aCART cell therapy) targeting any appropriate disease-associated antigen(e.g., an antigenic substance produced by cell affected by a particulardisease) within a mammal. Examples of disease-associated antigens thatcan be targeted by an adoptive T cell therapy provided herein include,without limitation desmopressin (associated with auto immune skindiseases). In another embodiment, the disease-associated antigens thatcan be targeted by an adoptive T cell therapy provided herein include,but are not limited to the DSG3 antigen, the B cell receptor (BCR) thatbinds to DSG3 in Pemphigus Vulgaris or the antigen MuSK. In a furtherembodiment, the disease-associated antigens that can be targeted by anadoptive T cell therapy provided herein include, but are not limited tothe BCR for MuSK in MuSK Myasthenia Gravis.

In some cases, one or more T cells having (e.g., engineered to have) areduced expression level of a cytokine polypeptide (e.g., a GM-CSF KO Tcells) used in an adoptive T cell therapy (e.g., a CART cell therapy)can be administered to a mammal having a cancer as a combination therapywith one or more additional agents used to treat a cancer. For example,one or more T cells having a reduced level of GM-CSF polypeptides usedin an adoptive cell therapy can be administered to a mammal incombination with one or more anti-cancer treatments (e.g., surgery,radiation therapy, chemotherapy (e.g., alkylating agents such asbusulfan), targeted therapies (e.g., GM-CSF inhibiting agents such aslenzilumab), hormonal therapy, angiogenesis inhibitors,immunosuppressants (e.g., interleukin-6 inhibiting agents such astocilizumab)) and/or one or more CRS treatments (e.g., ruxolitinib andibrutinib). In cases where one or more T cells having a reduced level ofGM-CSF polypeptides used in an adoptive cell therapy are used withadditional agents treat a cancer, the one or more additional agents canbe administered at the same time or independently. In some cases, one ormore T cells having a reduced level of GM-CSF polypeptides used in anadoptive cell therapy can be administered first, and the one or moreadditional agents administered second, or vice versa.

Lenzilumab (Humanigen, Burlingame, Calif.), an hGM-CSF neutralizingantibody in accordance with embodiments described herein and asdescribed in U.S. Pat. Nos. 8,168,183 and 9,017,674, each of which isincorporated herein by reference in its entirety, is a novel, first inclass Humaneered® monoclonal antibody that neutralizes human GM-CSF.

All scientific publications cited herein are hereby incorporated byreference in their entireties.

The following examples are presented in order to illustrate certainembodiments of the invention more fully. The examples should in no waybe construed, however, as limiting the broad scope of the inventiondescribed in the claims.

EXAMPLES Example 1 Generation of Cytokine to Deficient CART Cells toIncrease Therapeutic Index of CART Cell Therapy

This example describes the development of GM-CSF knocked out (GM-CSF KO)CART19 cells, and shows that the resulting GM-CSF KO CART19 cellsfunction normally and have enhanced expansion.

Experimental Design

CAR19 in B cell leukemia xenografts were used. These plasmids were usedfor packaging and lentivirus production as described herein. As a mousemodel, two models were employed:

1. Xenograft models: NSG mice were subcutaneously engrafted with theCD19 positive, luciferase positive cell line NALM6. Engraftment wasconfirmed by bioluminescence imaging. Mice were treated with human PBMCsintravenously and intra-tumor injection of lentivirus particles.Generation of CART cells is measured by flow cytometry. Trafficking ofCARTs to tumor sites is assessed and anti-tumor response is measured bybioluminescence imaging as a measure of disease burden.2. Humanized Immune System (HIS) mice from the Jackson Laboratory: Thesemice were injected with fetal CD34+ cells as neonates and thereforedevelop human hematopoiesis. We will engraft these mice with the CD19+cell line NALM6, as previously used. Similarly, we will generate CART19in vivo through the intratumoral injection of lentivirus particles. Thenwill measure the activity of CART19 cells in eradication of NALM6 andcompare that to ex vivo generated lenti-virally transduced CART19 cells(currently used in the clinic).

Materials and Methods: Generation of CAR Plasmid:

The anti-CD19 clone FMC63 was do novo synthesized into a CAR backboneusing 41BB and CD3 zeta and then cloned into a third generationlentivirus backbone.

To generate the control CART19 cells, normal donor T cells werenegatively selected using pan T cell kit and expanded ex vivo usinganti-CD3/CD28 Dynabeads (Invitrogen, added on the first day of culture).T cells were transduced with lentiviral supernatant one day followingstimulation at a multiplicity of infection (MOI) of 3. The anti-CD3/CD28Dynabeads were removed on day 6 and T cells were grown in T cell media(X-vivo 15 media, human serum 5%, penicillin, streptomycin andglutamine) for up to 15 days and then cryopreserved for futureexperiments. Prior to all experiments, T cells were thawed and restedovernight at 37° C.

Generation of GM-CSF Knock Out CART Cells:

GM-CSF knockout CART cells were generated with a CRISR-Cas9 system,using two methodologies:

1. gRNA was generated and cloned into a lentivirus vector that encodesCas9 and the gRNA. During T cell expansion, T cells were transduced withthis lentivirus on Day 1, on the same day and simultaneously with CAR19lentivirus particles. Cells were expanded for a period of 8 days andthen T cell were harvested, DNA isolated and sequenced to assess theefficiency of knockout. These cells were cryopreserved and used forfuture in vitro or in vivo experiments. A nucleic acid sequence encodingis shown in FIG. 5.2. mRNA was generated from the gRNA and used it to knock out GM-CSF. Todo so, gRNA was mixed with RNP at 1:1 ratio and then T cells wereelectroporated on Day 3 post stimulation with CD3/CD28 beads. Cells wereexpanded for a period of 8 days and then T cell were harvested, DNAisolated and sequenced to assess the efficiency of knockout. These cellswere cryopreserved and used for future in vitro or in vivo experiments

Cells

The NALM6 cell line was obtained from the ATCC and maintained in R10media (RPMI media, 10% fetal calf serum, penicillin, and streptomycin).NALM6-cells transduced with luciferase-GFP cells under the control ofthe EF1α promoter were used in some experiments as indicated.De-identified primary human ALL specimens were obtained from the MayoClinic Biobank. All samples were obtained after informed, writtenconsent. For all functional studies, cells were thawed at least 12 hoursbefore analysis and rested overnight at 37° C.

Flow Cytometry Analysis

Anti-human antibodies were purchased from BioLegend, eBioscience, or BDBiosciences. Cells were isolated from in vitro culture or from animals,washed once in PBS supplemented with 2% fetal calf serum, and stained at4° C. after blockade of Fc receptors. For cell number quantitation,Countbright beads (Invitrogen) were used according to the manufacturer'sinstructions (Invitrogen). In all analyses, the population of interestwas gated based on forward vs. side scatter characteristics followed bysinglet gating, and live cells were gated using Live Dead Aqua(Invitrogen). Surface expression of anti-CD19 CAR was detected bystaining with an Alexa Fluor 647-conjugated goat anti-mouse F(ab′)2antibody from Jackson Immunoresearch.

T Cell Function Assays: T Cell Degranulation and Intracellular CytokineAssays:

Briefly, T cells were incubated with target cells at a 1:5 ratio. Afterstaining for CAR expression; CD107a, CD28, CD49d and monensin were addedat the time of incubation. After 4 hours, cells were harvested andstained for CAR expression, CD3 and Live Dead staining (Invitrogen).Cells were fixed and permeabilized (FIX & PERM® Cell Fixation & CellPermeabilization Kit, Life technologies) and intracellular cytokinestaining was then performed.

Proliferation Assays:

T cells were washed and resuspended at 1×10⁷/ml in 100 μl of PBS andlabeled with 100 μl of CFSE 2.5 μM (Life Technologies) for 5 minutes at37° C. The reaction was then quenched with cold R10, and the cells werewashed three times. Targets were irradiated at a dose of 100 Gy. T cellswere incubated at a 1:1 ratio with irradiated target cells for 120hours. Cells were then harvested, stained for CD3, CAR and Live Deadaqua (Invitrogen), and Countbright beads (Invitrogen) were added priorto flow cytometric analysis.

Cytotoxicity Assays:

NALM6-Luc cells or CFSE (Invitrogen) labelled primary ALL samples wereused for cytotoxicity assay. In brief, targets were incubated at theindicated ratios with effector T cells for 4, 16, 24, 48, and/or 72hours. Killing was calculated either by bioluminescence imaging on aXenogen IVIS-200 Spectrum camera or by flow cytometry. For the latter,cells were harvested; Countbright beads and 7-AAD (Invitrogen) wereadded prior to analysis.

Residual Live Target Cells were CFSE+7-AAD-.

Secreted Cytokine Measurement:

Effector and target cells were incubated at a 1:1 ratio in T cell mediafor 24 or 72 hours as indicated. Supernatant was harvested and analyzedby 30-plex Luminex array according to the manufacturer's protocol(Invitrogen).

Results

GM-CSF KO CART cells were generated with a CRISR-Cas9 system. During Tcell expansion, T cells were transduced (Day 1) with lentivirus encodinggRNA and Cas9 and lentivirus encoding CARI 9. Cells were expanded for aperiod of 8 days. After 8 days, T cells were harvested, DNA wasisolated, and the isolated DNA was sequenced to assess the efficiency ofknockout. See, e.g., FIG. 1. T cells exhibited a knockout efficiency of24.1% (FIG. 2A), and CAR transduction efficiency was 73% (FIG. 2B).

To evaluate cell effector functions of GM-CSF KO CART cells, CART19,GM-CSF KO CART19, UTD, or GM-CSF KO UTD were co-cultured with the CD19positive cell line NALM6 at a ratio of 1:5. After 4 hours, the cellswere harvested, permeabilized, fixed, and stained for cytokines (FIG.3).

To evaluate proliferation of GM-CSF KO CART cells, expansion kineticswere followed after T cells were transduced. GM-CSF KO CART cells expandmore robustly than cells transduced with CART19 alone (FIG. 4).

These results demonstrate that GM-CSF knockout CARTs can enhance CARTcell function and antitumor activity. These results also demonstratethat blockade of GMCSF in combination with CART19 does not impact CARTcell effector functions.

Example 2 GM-CSF Depletion During CART Therapy Reduces Cytokine ReleaseSyndrome and Neurotoxicity and May Enhance CART Cell Function

This example investigates depleting granulocyte macrophagecolony-stimulating factor (GM-CSF) and myeloid cells as a potentialstrategy to manage CART cell associated toxicities. It was found thatthe GM-CSF blockade with a neutralizing antibody does not inhibit CARTfunction in vitro or in vivo. CART cell proliferation was enhanced invitro, and CART cells resulted in a more efficient control of leukemiain patient derived xenografts after GM-CSF depletion. Furthermore, in aprimary acute lymphoblastic leukemia xenograft model of CRS and NT,GM-CSF blockade resulted in a reduction of myeloid cell and T cellinfiltration in the brain, and ameliorated the development of CRS andNT. Finally, GM-CSF knocked out CART cells were generated throughCRISPR/cas9 disruption of GM-CSF during CART cell manufacturing.GM-CSF^(k/o) CART cells continued to function normally and had resultedin enhanced anti-tumor activity in vivo. These demonstrate that GM-CSFneutralization can abrogate neurotoxicity and CRS, and also can enhanceCART cell functions.

Materials and Methods Cells Lines and Primary Cells

NALM6 and MOLM13 were purchased from ATCC, Manassas, Va., USA,transduced with a luciferase-ZsGreen lentivirus (addgene) and sorted to100% purity. Cell lined were cultured in RIO (RPMI, 10% FCS v/v, 1% penstrep v/v). Primary cells were obtained from the Mayo Clinic biobank forpatients with acute leukemia under an institutional review boardapproved protocol. The use of recombinant DNA in the laboratory wasapproved by the Institutional Biosafety Committee (IBC).

Primary T Cells and CART Cells

Peripheral blood mononuclear cells (PBMC) were isolated fromde-identified donor blood apheresis cones using a FICOLL protocol (see,e.g., Dietz et al., 2006 Transfusion 46:2083-2089, which is incorporatedherein by reference in its entirety). T cells were separated withnegative selection magnetic beads (Stemcell technologies) and monocyteswere positively selected using CD14+ magnetic beads (Stemcelltechnologies). Primary cells were cultured in X-Vivo 15 media with 5%human serum, penicillin, streptomycin and glutamax. CD19 directed CARTcells were generated through the lentiviral transduction of normal donorT cells as described below. Second generation CARI 9 constructs were donova synthesized (IDT) and cloned into a third generation lentivirusunder the control of EF-1a promotor. The CD19 directed single chainvariable fragment was derived from the clone FMC63. A second generation41BB co-stimulated (FMC63-41BBz) CAR construct was synthesized and usedfor these experiments. Lentivirus particles were generated through thetransient transfection of plasmid into 293T virus producing cells, inthe presence of lipofectamine 3000, VSV-G and packaging plasmids. Tcells isolated from normal donors were stimulated using CD3/CD28stimulating beads (StemCell) at 1:3 ratio and then transduced withlentivirus particles 24 hours after stimulation at a multiplicity ofinfection of 3.0. Magnetic bead removal was performed on Day 6 and CARTcells were harvested and cryopreserved on Day 8 for future experiments.CART cells were thawed and rested in T cell medium 12 hours prior totheir use in experiments.

Generation of GM-CSF^(k/o) CART Cells:

A guide RNA (gRNA) targeting exon 3 of human GM-CSF was selected viascreening gRNAs previously reported to have high efficiency for humanGM-CSF.25 This gRNA was ordered in a CAS9 third generation lentivirusconstruct (lentiCRISPRv2), controlled under a U6 promotor (GenScript,Township, N.J., USA). Lentiviral particles encoding this construct wereproduced as described above. T cells were dual transduced with CAR19 andGM-CSFgRNA-lentiCRISPRv2 lentiviruses, 24 hours after stimulation withCD3/CD28 beads. CAR-T cell expansion was then continued as describedabove. To analyze efficiency of targeting GM-CSF, genomic DNA wasextracted from the GM-CSFk/o CART19 cells using PureLink Genomic DNAMini Kit (Invitrogen, Carlsbad, Calif., USA). The DNA of interest wasPCR amplified using Choice Taq Blue Mastermix (Thomas Scientific,Minneapolis, Minn., USA) and gel extracted using QIAquick Gel ExtractionKit (Qiagen, Germantown, Md., USA) to determine editing. PCR ampliconswere sent for Eurofins sequencing (Louisville, Ky., USA) and allelemodification frequency was calculated using TIDE (Tracking of Indels byDecomposition) software available at tide.nki.nl. FIGS. 15A-15B describethe gRNA sequence, primer sequences, and the schema for generation ofGM-CSFk/o CART 19 schema.

GM-CSF Neutralizing Antibodies and Isotype Controls

Lenzilumab (Humanigen, Brisbane, Calif.) is a humanized antibody thatneutralizes human GM-CSF, as described in U.S. Pat. Nos. 8,168,183 and9,017,674, each of which is incorporated herein by reference in itsentirety. For in vitro experiments, lenzilumab or isotype control 10ug/mL was used. For in vivo experiments, 10 mg/kg of lenzilumab orisotype control was injected, and the schedule, route and frequency areindicated in the individual experimental schema. In some experiments,anti-mouse GM-CSF neutralizing antibody (10 mg/kg) was also used, asindicated in the experimental schema.

T Cell Functional Experiments

Cytokine assays were performed 24 or 72 hours after a co-culture of CARTcells with their targets at 1:1 ratio as indicated. Human GM-CSFsingleplex (Millipore), 30-plex human multiplex (Millipore), or 30-plexmouse multiplex (Millipore) was performed on supernatant collected fromthese experiments, as indicated. This was analyzed using flow cytometrybead assay or Luminex, Intracellular cytokine analysis and T celldegranulation assays were performed following incubation of CART cellswith targets at 1:5 ratio for 4 hours at 37° C., in the presence ofmonensin, hCD49d, and hCD28. After 4 hours, cells were harvested andintracellular staining was performed after surface staining, followed byfixation and permealization (FIX & PERM Cell Fixation & CellPermeabilization Kit, Life Technologies). For proliferation assays, CFSE(Life Technologies) labeled effector cells (CART19), and irradiatedtarget cells were co cultured at 1:1. In some experiments with CD14+monocytes was added to the co-culture at 1:1:1 ratio as indicated. Cellswere co-cultured for 3-5 days, as indicated in the specific experimentand then cells were harvested and surface staining with anti-hCD3 andlive/dead aqua was performed. PMA/ionomycin was used as a positivenon-specific stimulant of T cells, at different concentrations asindicated in the specific experiments. For killing assays, theCD19+Luciferase+ ALL cell line NALM6 or the CD19-Luciferase+ controlMOLM13 cells were incubated at the indicated ratios with effector Tcells for 24 or 48 hours as listed in the specific experiment. Killingwas calculated by bioluminescence imaging on a Xenogen IVIS-200 Spectrumcamera (PerkinElmer, Hopkinton, Mass., USA) as a measure of residuallive cells. Samples were treated with 1 μl D-luciferin (30 ug/mL) per100 μl sample volume, 10 minutes prior to imaging.

Multi-Parametric Flow Cytometry

Anti-human antibodies were purchased from Biolegend, eBioscience, or BDBiosciences.

Cells were isolated from in vitro culture or from peripheral blood ofanimals (after ACK lysis), washed twice in phosphate-buffered salinesupplemented with 2% fetal calf serum and stained at 4° C. For cellnumber quantitation, Countbright beads (Invitrogen) were used accordingto the manufacturer's instructions (Invitrogen). In all analyses, thepopulation of interest was gated based on forward vs side scattercharacteristics, followed by singlet gating, and live cells were gatedusing Live Dead Aqua (Invitrogen). Surface expression of CAR wasdetected by staining with a goat anti-mouse F(ab′)2 antibody. Flowcytometry was performed on a four-laser Canto II analyzer (BDBiosciences). All analyses were performed using FlowJo X10.0.7r2.

Xenogeneic Mouse Models

Male and female 8-12-week old NOD-SCID-IL2ry−/−(NSG) mice were bred andcared for within the Department of Comparative Medicine at the MayoClinic under a breeding protocol approved by the Institutional AnimalCare and Use Committee (IACUC). Mice were maintained in an animalbarrier spaces that is approved by the institutional Biosafety Committeefor BSL2+ level experiments.

NALM6 Cell Line Xenografts

The CD19+, luciferase+ ALL NALM6 cell line was used to establish ALLxenografts. These xenograft experiments were approved by a differentIACUC protocol. Here, 1×10⁶ cells were injected intravenously via a tailvein injection. After injection, mice underwent bioluminescent imagingusing a Xenogen IVIS-200 Spectrum camera six days later, to confirmengraftment. Imaging was performed after the intraperitoneal injectionof 10 μl/g D luciferin (15 mg/ml). Mice were then randomized based ontheir bioluminescent imaging to receive different treatments as outlinedin the specific experiments. Typically, 1-2×10⁶ CART cells or UTD cellsare injected and exact doses are listed in the specific experimentaldetails. Weekly imaging was performed to assess and follow diseaseburden. Tail vein bleeding was done 7-10 days after injection of CARTcells to assess T cell expansion and as needed following that. Mouseperipheral blood was lysed using ACK lysing buffer (Thermofisher) andthen used for flow cytometry studies. Bioluminescent images wereacquired using a Xenogen IVIS-200 Spectrum camera (PerkinElmer,Hopkinton, Mass., USA) and analyzed using Living Image version 4.4(Caliper LifeSciences, PerkinElmer). For antibody treated mice, antibodytherapy (10 mg/kg lenzilumab or isotype control) was commenced IP, for atotal of 10 days.

Primary Patient Derived ALL Xenografts

To establish primary ALL xenografts, NSG mice first received 30 mg/kgbusulfan IP. The following day, mice were injected with 2×10⁶ primaryblasts derived from the peripheral blood of patients with relapsedrefractory ALL. Mice were monitored for engraftment for 4-6 weeks andwhen CD19+ cells were consistently observed in the blood (>1 cell/μl),they were randomized to receive different treatments of CART19 or UTD(1×10⁶ cells) with or without antibody therapy (10 mg/kg lenzilumab orisotype control IP for a total of 10 days, starting on the day theyreceived CART cell therapy). Mice were periodically monitored forleukemic burden via tail vein bleeding.

Primary Patient Derived ALL Xenografts for CRS/NT

Similar to the experiments above, mice were IP injected with 30 mg/kgbusulfan. The following day, they received 1-2×10⁶ primary blastsderived from the peripheral blood of patients with relapsed refractoryALL. Mice were monitored for engraftment for 4-6 weeks and when CD19+cell level was high (≥10 cells/μl), they received CART19 (2-5×10⁶ cells)and commenced antibody therapy for a total of 10 days, as indicated inthe details of the specific experiment. Mice were weighed on daily basisas a measure of their well-being. Brian MRI of the mice was performed5-6 days post CART injection and tail vein bleeding was performed 4-11days post CART injection. Brain MRI images were analyzed using Azalyze.

MRI Acquisition

A Bruker Avance II 7 Tesla vertical bore small animal MRI system (BrukerBiospin) was used for image acquisition to evaluate central nervoussystem (CNS) vascular permeability. Inhalation anesthesia was inducedand maintained via 3 to 4% isoflurane. Respiratory rate was monitoredduring the acquisition sessions using an MRI compatible vital signmonitoring system (Model 1030; SA Instruments, Stony Brook, N.Y.). Micewere given an IP injection of gadolinium using weight-based dosing of100 mg/kg, and after a standard delay of 15 min, a volume acquisitionT1-weighted spin echo sequence was used (repetition time=150 ms, echotime=8 ms, field of view: 32 mm×19.2 mm×19.2 mm, matrix: 160×96×96;number of averages=1) to obtain T1-weighted images. Gadolinium-enhancedMRI changes were indicative of blood-brain-barrier disruption.Volumetric analysis was performed using Analyze Software packagedeveloped by the Biomedical Imaging Resource at Mayo Clinic.

RNA-Seq on Mouse Brain Tissue

RNA was isolated using miRNeasy Micro kit (Qiagen, Gaithersburg, Md.,USA) and treated with RNase-Free DNase Set (Qiagen, Gaithersburg, Md.,USA). RNA-seq was performed on an Illumina HTSeq 4000 (Illumina, SanDiego, Calif., USA) by the Genome Analysis Core at Mayo Clinic. Thebinary base call data was converted to fastq using Illumina bcl2fastqsoftware. The adapter sequences were removed using Trimmomatic, andFastQC was used to check for quality. The latest human (GRCh38) andmouse (GRCm38) reference genomes were downloaded from NCBI. Genome indexfiles were generated using STAR, and the paired end reads were mapped tothe genome for each condition. HTSeq3 l was used to generate expressioncounts for each gene, and DeSeq2 was used to calculate differentialexpression. Gene ontology was assessed using Enrichr. FIG. 16 summarizesthe steps detailed above. RNA sequencing data are available at the GeneExpression Omnibus under accession number GSE121591.

Statistics

Prism Graph Pad and Microsoft Excel used to analyze data. The highcytokine concentrations in the heat map were normalized to “1” and lowconcentrations normalized to “O” via Prism. Statistical tests describedin figure legends.

Results

GM-CSF Neutralization In Vitro Enhances CAR-T Cell Proliferation in thePresence of Monocytes and does not Impair CAR-T Cell Effector Function.

If GM-CSF neutralization after CAR-T cell therapy is to be utilized as astrategy to prevent CRS and NT, it must not inhibit CAR-T cell efficacy.Therefore, our initial experiments aimed to investigate the impact ofGM-CSF neutralization on CAR-T cell effector functions. Here, CART19cells were co-cultured with or without the CD19+ ALL cell line NALM6 inthe presence of lenzilumab (GM-CSF neutralizing antibody) or an isotypecontrol (IgG). We established that lenzilumab, but not IgG controlantibody, was indeed able to completely neutralize GM-CSF (FIG. 6A) butdid not inhibit CAR-T cell antigen specific proliferation (FIG. 6B).When CART19 cells were co-cultured with the CD19+ cell line NALM6 in thepresence of monocytes, lenzilumab in combination with CART19demonstrated an exponential increase in antigen specific CART19proliferation compared to CART19 plus isotype control IgG (P<0.0001,FIG. 6C). To investigate CAR-T specific cytotoxicity, either CART19 orcontrol UTD T cells were cultured with the luciferase+CD19+NALM6 cellline and treated with either isotype control antibody or GM-CSFneutralizing antibody (FIG. 6D). GM-CSF neutralizing antibody treatmentdid not inhibit the ability of CAR-T cells to kill NALM6 target cells(FIG. 6D). Overall, these results indicate that lenzilumab does notinhibit CAR-T cell function in vitro and enhances CART19 cellproliferation in the presence of monocytes, suggesting that GM-CSFneutralization may improve CAR-T cell mediated efficacy.

GM-CSF Neutralization In Vivo Enhances CAR-T Cell Anti-Tumor Activity inXenograft Models.

To confirm that GM-CSF depletion does not inhibit CART19 effectorfunctions, we investigated the role of GM-CSF neutralization withlenzilumab on CART19 antitumor activity in xenograft models. First, arelapse model intended to vigorously investigate whether the antitumoractivity of CART19 cells was impacted by GM-CSF neutralization was used.NSG mice were injected with 1×10⁶ luciferase+NALM6 cells and then imaged6 days later, allowing sufficient time for mice to achieve very hightumor burdens. Mice were randomized to receive a single injection ofeither CART19 or UTD cells and 10 days of either isotype controlantibody or lenzilumab (FIG. 7A). GM-CSF assay on serum collected 8 daysafter CART19 injection revealed that lenzilumab successfully neutralizesGM-CSF in the context of CART19 therapy (FIG. 7B). Bioluminescenceimaging one week after CART19 injection showed that CART19 incombination with lenzilumab effectively controlled leukemia in this hightumor burden relapse model and significantly better than control UTDcells (FIG. 7C). Treatment with CART19 in combination with lenzilumabresulted in potent anti-tumor activity and improved overall survival,similar to CART19 with control antibody despite neutralization of GM-CSFlevels, indicating that GM-CSF does not impair CAR-T cell activity invivo (FIG. 8). Second, these experiments were performed in a primary ALLpatient derived xenograft model, in the presence of human PBMCs as thisrepresents a more relevant heterogeneous model. After conditioningchemotherapy with busulfan, mice were injected with blasts derived frompatients with relapsed ALL. Mice were monitored for engraftment forseveral weeks through serial tail vein bleedings and when the CD19+blasts in the blood were ≥1 μL, mice were randomized to receive CART19or UTD treatment in combination with PBMCs with either lenzilumab plusan anti-mouse GM-CSF neutralization antibody or isotype control IgGantibodies starting on the day of CART19 injection for 10 days (FIG.7D). In this primary ALL xenograft model, GM-CSF neutralization incombination with CART19 therapy resulted in a significant improvement inleukemic disease control sustained over time for more than 35 days postCART19 administration as compared to CART19 plus isotype control (FIG.7E). This suggests that GM-CSF neutralization may play a role inreducing relapses and increasing durable complete responses after CART19cell therapy.

GM-CSF CRISPR Knockout CAR-T Cells Exhibit Reduced Expression of GM-CSF,Similar Levels of Key Cytokines, and Enhanced Anti-Tumor Activity.

To confidently exclude any role for GM-CSF critical in CAR-T cellfunction, we disrupted the GM-CSF gene during CAR-T cell manufacturingusing a gRNA that has been reported to yield high efficiency, clonedinto a CRISPR lentivirus backbone. Using this gRNA, we achieved around60% knockout efficiency in CART19 cells (FIG. 9). When CAR-T cells werestimulated with the CD19+ cell line NALM6, GM-CSF^(k/o) CAR-T cellsproduced statistically significantly less GM-CSF compared to CART19 witha wild-type GM-CSF locus (“wild type CART19 cells”). GM-CSF knockout inCAR-T cells did not impair the production of other key T cell cytokines,including IFN-γ, IL-2, or CAR-T cell antigen specific degranulation(CD107a) (FIG. 10A) but did exhibit reduced expression of GM-CSF (FIG.10B). To confirm that GM-CSF^(k/o) CAR-T cells continue to exhibitnormal functions, we tested their in vivo efficacy in the high tumorburden relapsing xenograft model of ALL (as described in FIG. 7A). Inthis xenograft model, utilization of GM-CSF^(k/o) CART19 instead of wildtype CART19 markedly reduced serum levels of human GM-CSF at 7 daysafter CART19 treatment (FIG. 10B). Bioluminescence imaging data impliedthat GM-CSF^(k/o) CART19 cells show enhanced leukemic control comparedto CART19 in this model (FIG. 11). Importantly, GM-CSF^(k/o) CART19cells demonstrated significant improvement in overall survival comparedto wild type CART19 cells (FIG. 10C). Other than GM-CSF, nostatistically significantly alterations in either human (FIG. 10D) ormouse (FIG. 10E) cytokines were detected. Together, these resultsconfirm FIGS. 6 and 7, indicating that GM-CSF depletion does not impaircytokines that are critical to CAR-T efficacy functions. In addition,the results in FIG. 10 indicate that GM-CSFk/o CART may represent atherapeutic option for “built in” GM-CSF control as a modificationduring CAR-T cell manufacturing.

Patient Derived Xenograft Model for Neurotoxicity and Cytokine ReleaseSyndrome

In this model, conditioned NSG mice were engrafted with primary ALLblasts and monitored for engraftment for several weeks until theydeveloped high disease burden (FIG. 12A). When the level of CD19+ blastsin the peripheral blood was ≥10/μL, mice were randomized to receivedifferent treatments as indicated (FIG. 12A). Treatment with CART19(with control IgG antibodies or with GM-CSF neutralizing antibodies)successfully eradicated the disease (FIG. 12B). Within 4-6 days aftertreatment with CART19, mice began to develop motor weakness, hunchedbodies, and progressive weight loss; symptoms consistent with CRS andNT. This was associated with elevation of key serum cytokines 4-11 dayspost CART19 injection similar to what is seen in human CRS after CAR-Tcell therapy (including human GM-CSF, TNF-α, IFN-γ, IL-10, IL-12, IL-13,IL-2, IL-3, IP-10, MDC, MCP-1, MIP-1a, MIP-1, and mouse IL-6, GM-CSF,IL-4, IL-9, IP-10, MCP-1, and MIG). These mice treated with CART19 alsodeveloped NT as indicated by brain MRI analyses revealing abnormal T1enhancement, suggestive of blood-brain barrier disruption and possiblybrain edema (FIG. 12D), together with flow cytometric analysis of theharvested brains revealing infiltration of human CART19 cells (FIG.12E). In addition, RNA-seq analyses of brain sections harvested frommice that developed these signs of NT showed significant upregulation ofgenes regulating the T cell receptor, cytokine receptors, T cell immuneactivation, T cell trafficking, and T cell and myeloid celldifferentiation (Table 1).

TABLE 1 Table of canonical pathways altered in brains from patientderived xenografts after treatment with CART19 cells. Adj CanonicalPathway P-Value Genes regulation of immune 9.45E−14 IFITM1, ITGB2, TRAC,ICAM3, CD3G, PTPN22, CD3E, response (G0:0050776) ITGAL, SAMHD1, SLA2,CD3D, ITGB7, SLAMF6, B2M, NPDC1, CD96, BTN3A1, ITGA4, SH2D1A, HLA-B,HLA-C, BTN3A2, HLA-A, CD8B, SELL, CD8A, CD226, CD247, CLEC2D, HCST,B1RC3 cytokine-mediated 1.36E−12 IFITM1, SP100, TRADD, ITGB2, IL2RG,SAMHD1, IL27RA, signaling pathway OASL, CNN2, IL18RAP, RIPK1, CCR5,IL12RB1, B2M, (G0:0019221) GBP1, IL6R, JAK3, CCR2, IL32, ANXA1, IL4R,TGFB1, IL10RB, IL10RA, STAT2, PRKCD, HLA-B, HLA-C, IL16, HLA-A,TNFRSF1B, CD4, IRF3, OAS2, IL2RB, FAS, TNFRSF25, LCP1, P4HB, IL7R,MAP3K14, CD44, IL18R1, IRF9, MYD88, B1RC3 T cell receptor complex1.30E−11 ZAP70, CD4, CD6, CD8B, CD8A, CD3G, CD247, CD3E, (G0:0042101)CD3D, CARD11 T cell activation 2.07E−11 ITK, RHOH, CD3G, NLRC3, PTPN22,CD3E, SLA2, CD3D, (G0:0042110) CO2, ZAP70, CD4, PTPRC, CD8B, CD8A, LCK,CD28, LCP1, LAT regulation of T cell 2.46E−10 PTPN22, LAX1, CCDC88B,CD2, CD4, LCK, SIT1, TBX21, activation (G0:0050863) TIGIT, JAK3, LAT,PAG1, CCR2 T cell receptor signaling 4.35E−08 ITK, BTN3A1, TRAC, WAS,CD3G, PTPN22, BTN3A2, CD3E, pathway (G0:0050852) CD3D, ZAP70, CD4,PTPRC, LCK, GRAP2, LCP2, CD247, CARD11, LAT, PAG1 positive regulation of1.57502E−07   GBP5, ANXA1, TGFB1, CYBA, PTPN22, PARK7, TMEM173, cytokineproduction CCDC88B, MAVS, CD6, IRF3, CD28, RIPK1, SLAMF6, (G0:0001819)CD46, IL12RB1, TIGIT, IL6R, CARD11, MYD88, CCR2 T cell differentiation2.36E−07 ZAP70, CD4, ANXA1, PTPRC, CD8A, LCK, CD28, RHOH, (G0:0030217)PTPN22, CD3D cytokine receptor activity 2.43E−07 IL4R, IL10RB, IL10RA,IL2RG, CD4, CXCR3, IL2RB, (G0:0004896) CCR5, IL12RB1, IL7R, IL6R, CD44,CCR2 type I interferon 3.27E−07 IFITM1, SP100, IRF3, OAS2, STAT2, HLA-B,HLA-C, signaling pathway HLA-A, SAMHD1, IRF9, MYD88, OASL (G0:0060337)response to cytokine 0.0004679 SIGIRR, IFITM1, SP100, HCLS1, RIPK1,PTPN7, IKBKE, (G0:0034097) IL6R, JAK3, IL18R1, MYD88, AES regulation ofinnate 0.001452  GBP5, GFI1, STAT2, ADAM8, NLRC3, PTPN22, SAMHD1, immuneresponse B1RC3 (G0:0045088) regulation of tumor 0.003843  CD2, MAVS,CYBA, NLRC3, PTPN22, RIPK1, SLAMF1 necrosis factor production(G0:0032680) T cell receptor binding 0.0102397 LCK, CD3G, CD3E(G0:0042608) regulation of tumor 0.0124059 SHARPIN, TRADD, CASP4, RIPK1,TRAF1, B1RC3 necrosis factor-mediated signaling pathway (G0:0010803)positive regulation of 0.0376647 CD4, HCLS1, RIPK1, EV12B myeloidleukocyte differentiation (G0:0002763)GM-CSF Neutralization In Vivo Ameliorates Cytokine Release Syndrome andNeurotoxicity after CART19 Therapy in a Xenograft Model.

Using the xenograft patient derived model for NT and CRS shown in FIG.4A, we investigated the effect of GM-CSF neutralization on CART19toxicities. To rule out the cofounding effect of mouse GM-CSF, micereceived CART19 cells in combination with 10 days of GM-CSF antibodytherapy (10 mg/kg lenzilumab and 10 mg/kg anti-mouse GM-CSF neutralizingantibody) or isotype control antibodies. GM-CSF neutralizing antibodytherapy prevented CRS induced weight loss after CART19 therapy (FIG.13A). Cytokine analysis 11 days after CART19 cell therapy showed thathuman GM-CSF was neutralized by the antibody (FIG. 13B). In addition,GM-CSF neutralization resulted in significant reduction of several human(IP-10, IL-3, IL-2, IL-1Ra, IL-12p40, VEGF, GM-CSF) (FIG. 5C) and mouse(MIG, MCP-1, KC, IP-10) (FIG. 13D) cytokines. Interferon gamma-inducedprotein (IP-10, CXCL1O) is produced by monocytes among other cell typesand serves as a chemoattractant for numerous cell types includingmonocytes, macrophages, and T cells. IL-3 plays a role in myeloidprogenitor differentiation. IL-2 is a key T cell cytokine. Interleukin-1receptor antagonist (IL-1Ra) inhibits IL-1. (IL-1 is produced bymacrophages and is a family of critical inflammatory cytokines.)IL-12p40 is a subunit of IL-12, which is produced by macrophages amongother cell types and can encourage Th1 differentiation. Vascularendothelial growth factor (VEGF) encourages blood vessel formation.Monokine induced by gamma interferon (MIG, CXCL9) is a T cell chemoattractant. Monocyte chemoattractant protein 1 (MCP-1, CCL2) attractsmonocytes, T cells, and dendritic cells. KC (CXCL1) is produced bymacrophages among other cell types and attracts myeloid cells such asneutrophils. There was also a trend in reduction of several other humanand mouse cytokines after GM-CSF neutralization. This suggests thatGM-CSF plays a role in the downstream activity of several cytokines thatare instrumental in the cascade that results in CRS and NT.

Brain MRIs 5 days after CAR19 treatment showed that GM-CSFneutralization reduced T1 enhancement as a measure of braininflammation, blood-brain barrier disruption, and possibly edema,compared to CART19 plus control antibodies. The MRI images after GM-CSFneutralization (with lenzilumab and anti-mouse GM-CSF antibody) weresimilar to baseline pre-treatment scans, suggesting that GM-CSFneutralization effectively helped abrogated the NT associated withCART19 therapy (FIGS. 14A, 14B). Using human ALL blasts and human CART19in this patient-derived xenograft model, GM-CSF neutralization afterCART19 reduced neuro-inflammation by 75% compared to CART19 plus isotypecontrols (FIG. 14B). This is a significant finding, and the first timeit has been demonstrated in vivo that the NT caused by CART19 can beeffectively abrogated. Human CD3 T cells were present in the brain afterCART19 therapy as assayed by flow cytometry, and with GM-CSFneutralization, there was a trend toward reduction in brain CD3 T cells(FIG. 14C). Finally, a trend in reduction of CD11b+ bright macrophageswas observed in the brains of mice receiving GM-CSF neutralizationduring CAR-T cell therapy compared to isotype control during CAR-Ttherapy (FIG. 14D), implicating that GM-CSF neutralization helps reducemacrophages within the brain.

Example 3-A Combination Therapy with GM-CSF Gene KO in CART19 Cells(GM-CSF^(k/o) CART19) and hGM-CSF Neutralizing Antibody (Lenzilumab) forCAR19 T Derived GM-CSF

Lenzilumab (e.g., 10 mg/kg or up to 30 mg/kg or 1,800 mg flat dosing) isadministered to a subject in combination with GM-CSF^(k/o) CART19 cells.GM-CSF^(k/o) CAR-T cells help control GM-CSF release uponcontact/binding of CAR-T cells with tumor cells. The reduction of GM-CSFsecretion at the tumor site results in less activation and traffickingof inflammatory myeloid cells and reduced levels of MCP-1, IL-6, IP10,KC, MIP-1a, MIP-1b, MIG, VEGF, IL-1RA, and IL-12p40 in measuredsystemically. The reduced cytokine levels prevents or reduces theincidence or severity of CRS and NT. The addition of lenzilumab ensuresthat GM-CSF is neutralized from all sources and helps deplete MDSCs fromthe tumor microenvironment. Lenzilumab dosing can be repeated atintervals of every two weeks to insure continued depletion of MDSCs. Thecombination of GM-CSF^(k/o) CAR-T cells with lenzilumab results inimproved response rates, improved progression free survival, andimproved overall survival in patients treated with the combinationtherapy vs. control. The combination therapy also results in lowerlevels (or elimination) of the toxicities associated with CAR-T celltherapy, including CRS and NT.

Example 3-B Combination Therapy with GM-CSF Gene KO in CART19 Cells(GM-CSF^(k/o) CART19) and hGM-CSF Neutralizing Antibody (Lenzilumab) forCAR19 T Derived GM-CSF

Lenzilumab (/600-1800 mg) is administered to a subject in combinationwith GM-CSF^(k/o) CART19 cells.

Non-Hodgkins Lymphoma cancer patients are pre-conditioned prior totherapy. They are dosed I.V. with anti-hGM-CSF antibody (600-1800 mg)followed by 2×10⁶ transduced autologus CD19CART cells (GM-CSF^(KO)). Atspecific times after treatment effects are assessed e.g., safety, bloodchemistry, neurologic assessments, disease status. The treatment may berepeated on a monthly basis until there is no further detectable canceror there is a significant reduction in cancer load.

Example 4 Recombinant Anti-hGM-CSF Antibody, Lenzilumab, Reduces MyeloidCell Infiltration in the CNS

CD14+ cells comprise a greater proportion of the CNS cell population inhuman patients with grade 3 or above neurotoxicity, as shown in FIG.17A. Administration of recombinant anti-hGM-CSF antibody (lenzilumab)that binds to and neutralizes human GM-CSF to mice treated with CART19therapy demonstrated a reduction in CNS infiltration by CD14+ cells andby CD11b+ cells, as shown in FIG. 17B in comparison to untreated miceand mice treated only with CART19 therapy. A primary ALL mouse model wasused, as detailed below for the NT experiments.

Primary Patient Derived ALL Xenografts for CRS/NT

Similar to the experiments above, mice were IP injected with 30 mg/kgbusulfan. The following day, they received 1-2×10⁶ primary blastsderived from the peripheral blood of patients with relapsed refractoryALL. Mice were monitored for engraftment for 4-6 weeks and when CD19+cell level was high (≥10 cells/μl), they received CART19 (2-5×10⁶ cells)and commenced antibody therapy for a total of 10 days, as indicated inthe details of the specific experiment. (as described in Example 2).Mice were weighed on daily basis as a measure of their well-being. BrianMRI of the mice was performed 5-6 days post CART injection and tail veinbleeding was performed 4-11 days post CART injection. Brain MRI imageswere analyzed using Azalyze.

Example 5 Gene Editing Technologies to Knockout GM-CSF Genes in T Cells

Several strategies are being pursued by various groups to incorporategene editing into the development of next-generation chimeric antigenreceptor (CAR) T cells for the treatment of various cancers. Severetoxicity (cytokine release syndrome and neurotoxicity) is associatedwith CAR T cell therapy and can result in poor patient outcomes. A keyinitiator in the toxicity process seems to be CART cell derived GM-CSF.

Gene-editing (with e.g., engineered nucleases) may be used to KO GM-CSFgenes in T cells and/or gene/s encoding proteins essential for GM-CSFgene expression. Nucleases useful for such genome editing include,without limitation, CRISPR-associated (Cas) nucleases, zinc-fingernucleases (ZFNs), transcription activator-like effector (TALE)nucleases, and homing endonucleases (HEs) also known as meganucleases

Zinc-Finger Nuclease use for GM-CSF

A GM-CSF gene in CART cells can be inactivated using Zinc FingerNuclease (ZFN) technology. DNA sequence specific nucleases cleave theGM-CSF gene/s and DNA double strand break repair results in inactivationof the gene/s. The sequence specific nucleases are created by combiningsequence specific DNA binding domains (Zinc fingers) with a Fok1endonuclease domain. The targeted nuclease acts as a dimer and twodifferent DNA recognition domains are employed to provide site specificcleavage. Engineering of the Fok1 endonuclease ensures that heterodimersform rather than homodimers. Thus, the obligate heterodimer Fok1-ELvariant provides a higher level of specificity.

Clinical experience to date with gene KO approaches using ZFN technologyis limited. However, in a small safety study in which the CCR5 receptorwas knocked-out using ZFN technology and the T-cells re-introduced intoHIV patients, there was a notable survival advantage of the modified Tcells vs unmodified when anti-retroviral drug therapy was stopped.

The best effect was observed when biallelic gene disruption wasachieved. This suggests that the KO technology that achieves greatest %gene disruption is likely to be the most effective (Singh 2017, Tebas2014). In some human cell types biallelic targeting efficiency isincreased by RAD51 over expression and valproic acid treatment (Takayama2017).

Exons 1-4 of the human GM-CSF gene can be targeted with ZFNs that formpairs within the chosen target region. A potential advantage totargeting close to the translational initiation codon within the DNAsequence is that it ensures that the gene knockout does not result in alarge fragment of protein that is still synthesized. Such proteinfragments could have unwanted biological activities.

A variety of tools are available for the identification of potentialzinc finger nuclease (ZFN) sites in specific target sequences. Anexample of such tools can be found at:http://bindr.gdcb.iastate.edu/ZiFiT/. Vectors for the expression ofpairs of ZFNs identified in this way (for use in GM-CSF gene KO) aretested in human cells expressing GM-CSF and the effectiveness of genedisruption for each pair is measured by changes in GM-CSF productionwithin a pool of cells. Pairs of ZFNs demonstrating the highestreduction in GM-CSF levels are chosen for testing in human CART cells.

For example, autologous T-cells can be transduced ex vivo with areplication deficient recombinant Ad5 viral vector encoding pairs of theGM-CSF specific ZFNs, resulting in modification of the GM-CSF gene. Thevector supports only transient expression of genes encoded by thevector. The two ZFNs bind to a composite bp sequence found specificallyin the region chosen for mutagenesis (within exons 1, 2, 3 or 4) of theGM-CSF gene. Expression of the GM-CSF-specific ZFNs induces a doublestranded break in the cellular DNA which is repaired by cellularmachinery leading to random sequence insertions or deletions in thetransduced cells. These insertions and deletions disrupt the GM-CSFcoding sequence leading to frameshift mutation and termination ofprotein expression.

The T Cell Manufacture/Patient-Specific Sample

Study subjects undergo a 10 liter leukapheresis to collect >10⁹ whiteblood cells. The leukapheresis product is enriched for CD4+ cells bydepleting monocytes via counterflow centrifugal elutriation, and bymagnetically depleting CD8+ T-cells, both employing a single-useclosed-system disposable set. The resulting enriched CD4+ T-cells areactivated with anti-CD3/anti-CD28 mAb coated paramagnetic beads andtransduced with vector encoding CAR T and vector encoding ZFNs. Cellsare then expanded and cultured in a closed system. T-cell expansioncontinues after transfer to a WAVE Bioreactor for additional expansionunder perfusion conditions. At the end of the culture period, cells aredepleted of magnetic beads, washed, concentrated, and cryopreserved.

Primary T cells may also be treated with treated with other agents,e.g., valproic acid in order to increase bi-allelic targeting efficiencyof the ZFNs.

Putative Targeting Sequences Exon 1 (SEQ ID NO: 14)ATG TGG CTG CAG AGC CTG CTG CTC TCG GGC (SEQ ID NO: 15)TAC ACC GAC GTC TCG GAC GAC GAG AGC CCG (SEQ ID NO: 14 continued)CTC GCC CAG CCC CAG CAC GCA GCC (SEQ ID NO: 15 continued)GAG CGG GTC GGG GTC GTG CGT CGG Exon 2 (SEQ ID NO: 16)AAT GAA ACA GTA GAA GTC ATC TCA GAA ATG (SEQ ID NO: 17)TTA CTT TGT CAT CTT CAG TAG AGT CTT TAC (SEQ ID NO: 16 continued)GAA GTC ATC TCA GAA ATG TTT GAC (SEQ ID NO: 17 continued)CTT CAG TAG AGT CTT TAC AAA CTG Design Exon 3 (SEQ ID NO: 18)GAG CCG A CC TGC CTA CAG ACC CGC CTG GAG (SEQ ID NO: 19)CTC GGC TGG ACG GAT GTC TGG GCG GAC CTC (SEQ ID NO: 18 continued)GCC TAC AGA CCCGCCT GGA GCT GTA (SEQ ID NO: 19 continued)CGG ATG TCT GGGCGGA CCT CGA CAT Exon 4 (SEQ ID NO: 20)GAA ACT TCC TGT GCA ACC CAG ATT ATC ACC (SEQ ID NO: 21)CTT TGA AGG ACA CGT TGG GTC TAA TAG TGG (SEQ ID NO: 20 continued)TGC AAC CCA GAT TATC ACC TTT GAA (SEQ ID NO: 21 continued)ACG TTG GGT CTA ATAG TGG AAA CTT

TALENS

GM-CSF gene/s in T cells can also be inactivated using activator-likeeffector nucleases (TALENS). TALENS are similar to ZFNs in that theycomprise a Fok1 nuclease domain fused to a sequence specific DNA-bindingdomain. The targeted nuclease then makes a double-strand break in theDNA and error-prone repair creates a mutated target gene. TALENS can beeasily designed using a simple protein-DNA code that uses DNA bindingTALE (transcriptional-activator—like effectors) repeat domains toindividual bases in a binding site. The robustness of TALEN means thatgenome editing is a reliable and facile process (Reyon D., et al., 2012Nat Biotechnol. 2012 May; 30(5):460-5. doi: 10.1038/nbt.2170, which isincorporated herein by reference its entirety.)

By way of examples, some TALE target sequences within Exon 1 of humanGM-CSF gene are:

1. (SEQ ID NO: 22) TGGCTGCAGAGCCTGCTG CTCTTGGGCACTGTGG CCTGCAGCATCTCTGCA2. (SEQ ID NO: 23) TTGGGCACTGTGGCCTGC AGCATCTCTGCACCCG CCCGCTCGCCCAGCCCCA Examples of TALE target sequences in Exon 4 of human GM-CSF gene: 1.(SEQ ID NO: 24) TGTGCAACCCAGATTATC ACCTTTGAAAGTTTCA AAGAGAACCTGAAGGA 2.(SEQ ID NO: 25) TCCTGTGCAACCCAGATT ATCACCTTTGAAAGTT TCAAAGAGAACCTGAA 3.(SEQ ID NO: 26) TTATCACCTTTGAAAG TTTCAAAGAGAACCTGA AGGACTTTCTGCTTGTCA

CRISPR Cas-9 Mediated GM-CSF Gene KO in Primary T-Cells.

The CRISPR (clustered regularly interspaced short palindromic repeats),Cas-9 system is composed of Cas9, a RNA-guided nuclease and a shortguide RNA (gRNA) that facilitates the generation of site-specific DNAbreaks, which are repaired by cell-endogenous mechanisms. Cas9/gRNA RNPdelivery to primary human T-cells results in highly efficient targetgene modification. CRISPR/Cas9 mediated methods to knockout the GM-CSFgene are described by Detailed protocols see Oh, S. A., Seki, A., &Rutz, S. (2018) Current Protocols in Immunology, 124, e69. doi:10.1002/cpim.69, and Seki and Rutz, J Exp. Med. 2018 Vol. 215 No. 3985-997, each of which is incorporated herein by reference its entirety.

GM-CSF inactivation by gene KO has been reported to reduce cytokinerelease syndrome and neurotoxicity and improve anti-tumor activity inCAR T treated mice with tumor xenografts (as described by Sterner R M etal., 2018 Blood 2018:blood-2018-10-881722; doi:https://doi.org/10.1182/blood-2018-10-881722), which is incorporatedherein by reference its entirety.

Inactivation of GM-CSF Gene by CRISPR Approach Targeting Exon 1 or 2 or3 or 4.

Multiple, e.g., 3 Cas9 constructs targeting 3 different sequences withinthe GM-CSF gene may be used so as to ensure efficient gene inactivationin all samples. (This is easily done with CRISPR compared to other geneediting methods.)

High frequency of bi-allelic KO reported using Cas9 (as described byZhang, Y., et al. Methods. 2014 September; 69(2): 171-178.doi:10.1016/j.ymeth.2014.05.003, which is incorporated herein byreference its entirety. This high frequency of bi-allelic KO provides apossible advantage.

Other Gene Silencing Technologies for GM-CSF KO in CAR T Cells

Other methods that can be used for gene silencing are well known tothose ordinary skilled in the art and may include, without limitation,homing endonucleases (HEs) also known as meganucleases, RNA interference(RNAi), short interfering RNS (siRNA), DNA-directed RNA interference(ddRNAi).

Combination of GM-CSF Gene KO in CAR T Cells and Neutralizing Antibodyfor Non-CAR T Derived GM-CSF.

Removal/neutralization of all GM-CSF in patients requires anti-GM-CSFantibody, anti-receptor antibody, or soluble receptor-Fc fusion used incombination with GM-CSF gene KO in CART cells. The CART cellsadministered, include but are not limited to, GM-CSF^(k/o) CART cells.In one embodiment, the administered GM-CSF^(k/o) CART cells areGM-CSF^(k/o) CART19.

An anti-GM-CSF neutralizing antibody is administered in this combinationtherapy, including but not limited to Lenzilumab. Lenzilumab is a novel,high affinity, recombinant human, neutralizing anti-hGM-CSF antibody.Studies in non-human primates have shown that this antibody is safe whenrepeat-dosed, even at doses as high as >100 mg/kg/wk. for 6 weeks. Thisantibody is also safe in humans when repeat-dosed (7 doses of 400mg/dose, over 24 weeks to severe asthmatics). This antibody can be usedin combination with GM-CSF KO CART cell therapy in cancer patientsproviding complete neutralization of human GM-CSF. Cancer patients aredosed I.V. with anti-hGM-CSF antibody (600-1800 mg) followed by 2×10⁶CAR T cells (GM-CSF^(KO)). At specific times after treatment effects areassessed, e.g., safety, blood chemistry, neurologic assessments, diseasestatus. The treatment may be repeated on a monthly or 3 monthly basisand may result in disease remission and improved progression freesurvival.

GM-CSF can also be neutralized using an anti-human GM-CSF receptor alpha(RU) antibody (as described in Minter, R R, et al. 2012DOI:10.1111/j.1476-5381.2012.02173.x). Cancer patients are dosed I.V.with anti-hGM-CSF receptor antibody (70-700 mg) followed by 2×10⁶ CAR Tcells (GM-CSF^(KO)). At specific times after treatment effects areassessed, e.g., safety, blood chemistry, neurologic assessments, diseasestatus. Treatment results in disease remission and improved progressionfree survival. The treatment may be repeated on a monthly basis untilthere is no further detectable cancer or there is a significantreduction in cancer load.

Example 6 GM-CSF Disruption in CART Cells Ameliorates CART CellActivation and Reduces Activation-Induced Cell Death

It was hypothesized that GM-CSF depletion in CART cells results inreduced activation-induced cell death (AICD) and enhanced anti-tumoractivity independent of the effect on myeloid cell activation. In thepresent Example, how GM-CSF disruption in CART cells impacts theirfunctions was tested.

Methods

Cell lines. The acute lymphoblastic leukemia cell line NALM6 waspurchased from ATCC (CRL-3273, Manassas, Va., USA). Cell lines werecultured in R10 (RPMI 1640, Gibco, Gaithersburg, Md., US), 10% FetalBovine Serum (FBS, Millipore Sigma, Ontario, Canada), and 1%Penicillin-Streptomycin-Glutamine (Gibco, Gaithersburg, Md., US). Celllines are kept in culture after 20 passages, and fresh aliquots arethawed every 7-8 weeks. The use of recombinant DNA in the laboratory wasapproved by the Mayo Clinic Institutional Biosafety Committee (IBC).CART Cells. Generation of constructs, lentiviral production, titration,GM-CSF^(k/o) CART19 cells, and T cell functional experiments wereperformed as previously described in Sterner R M, Sakemura R, Cox M J,et al. GM-CSF inhibition reduces cytokine release syndrome andneuroinflammation but enhances CAR-T cell function in xenografts. Blood.2019; 133(7):697-709 and Sterner R M, Cox M J, Sakemura R, Kenderian SS. Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells. J Vis Exp.2019(149), each of which is incorporated herein by reference in itsentirety.

Flow Cytometric Analysis. Extracellular staining, acquisition, andgating were previously described in Sterner R M, et al. Blood. 2019;133(7):697-709, which is incorporated herein by reference in itsentirety. The following antibodies were used: anti-CD116 (GM-CSFRα)(clone 4H1) FITC (BioLegend, San Diego, Calif., USA), anti-CD131(GM-CSFRβ) (clone 1C1) PE (BioLegend, San Diego, Calif., USA), CD262(clone DJR2-4) PE (BioLegend, San Diego, Calif., USA), CD3 (clone OKT3)BV421 (BioLegend, San Diego, Calif., USA), CD3 (clone SK7) APC-H7 (BDPharmingen, San Jose, Calif., USA), CD4 (clone OKT4) FITC (eBioscience,San Diego, Calif., USA), CD8 (clone SKi) PerCP (BioLegend, San Diego,Calif., USA), CD3 BV650 (BioLegend, San Diego, Calif., USA), CD45 BV421(BioLegend, San Diego, Calif., USA), CD20 PE (BioLegend, San Diego,Calif., USA), CD25 PE-Cy7 (BioLegend, San Diego, Calif., USA), CD69BV785 (BioLegend, San Diego, Calif., USA), HlA-DR APC-Fire/750(BioLegend, San Diego, Calif., USA), and mCD45 PE (BioLegend, San Diego,Calif., USA). Absolute quantification was obtained using volumetricmeasurement.

Sequencing. RNA isolation and analysis were previously described inSterner R M, et al. Blood. 2019; 133(7):697-709, which is incorporatedherein by reference in its entirety. DNA was isolated using PureLinkGenomic DNA Mini Kit (Invitrogen, Carlsbad, Calif., USA), prepared withAgilent SureSelectXT (Santa Clara, Calif., USA), and sequenced onIllumina HiSeq 4000 (Illumina, San Diego, Calif., USA) by the MedicalGenome Facility Genome Analysis Core (Mayo Clinic, Rochester, Minn.,USA). Burrows-Wheeler Aligner, as described in Li H, Durbin R. Fast andaccurate long-read alignment with Burrows-Wheeler transform.Bioinformatics. 2010; 26(5):589-595, which is incorporated herein byreference in its entirety, and Genome Analysis Toolkit, as described inMcKenna A, Hanna M, Banks E, et al. The Genome Analysis Toolkit: aMapReduce framework for analyzing next-generation DNA sequencing data.Genome Res. 2010; 20(9):1297-1303, which is incorporated herein byreference in its entirety, were used to align reads to GRCh28 and callvariants. SAS 9.4 (SAS Institute Inc., Cary, N.C., USA) was used to finddifferences and filter by genomic prevalence (allele frequency ≤1%), asdescribed in Genomes Project C, Auton A, Brooks L D, et al. A globalreference for human genetic variation. Nature. 2015; 526(7571):68-74,which is incorporated herein by reference in its entirety. CRISPR/Cas9target online predictor (CCTop) off-target predictions werecross-referenced, as described in Stemmer M, et al. CCTop: An Intuitive,Flexible and Reliable CRISPR/Cas9 Target Prediction Tool. PLoS One.2015; 10(4):e0124633, which is incorporated herein by reference in itsentirety. Single nucleotide variants (SNVs) or insertions/deletions(indels) were compared between knockouts and controls. Statisticaltesting using Wilcoxon signed-rank test performed using GraphPad Prismversion 8.1.1 for Windows (GraphPad Software, La Jolla, Calif., USA,www.graphpad.com), as described in Iyer V, Boroviak K, Thomas M, et al.No unexpected CRISPR-Cas9 off-target activity revealed by triosequencing of gene-edited mice. PLoS Genet. 2018; 14(7):e1007503, whichis incorporated herein by reference in its entirety.

Data Sharing Statement. Sequencing data are available at BioProjectPRJNA623000.

Flow Cyometric Analysis. All anti-human and anti-mouse antibodies werepurchased from BioLegend, eBioscience or BD Biosciences (San Diego,Calif., USA). Cells were always washed twice in phosphate-bufferedsaline supplemented with 2% FBS (Millipore Sigma, Ontario, Canada) and1% sodium azide (Ricca Chemical, Arlington, Tex., USA). In all analyses,populations of interest were gated based on forward vs. side scattercharacteristics, followed by singlet gating, and live cells were gatedfollowing staining with LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit(Invitrogen, Carlsbad, Calif., USA). Surface expression of CAR wasdetected by staining with a goat anti-mouse F(ab′)2 antibody(Invitrogen, Carlsbad, Calif., USA). Expression of GM-CSF receptors aand P was detected with anti-human CD116 (4H1) FITC (305906, BioLegend,San Diego, Calif.) and anti-human CD131 (1C1) PE (306104, BioLegend, SanDiego, Calif.), respectively. Cytometric data were acquired using aCytoFLEX Flow Cytometer (Beckman Coulter, Chaska, Minn., USA). Gatingwas performed using Kaluza version 2.1 (Beckman Coulter, Chaska, Minn.,USA).

TUNEL assay. One million CART19 cells or GM-CSF^(k/o) CART19 at 1×10⁶/mLwere co-cultured with 1 million irradiated NALM6 at 0.5×10⁶/mL for 0, 1,2, and 4 hours. To assess these samples with the TUNEL assay, themanufacturer's protocol was followed (APO-BRDU MilliporeSigma, St.Louis, Mo., USA). Briefly, the cells were fixed at each timepoint andstored at −20° C. To measure apoptosis, the cells were then washed andincubated with DNA labeling solution containing TdT and Br-dUTP. Thenthe cells were labeled with anti-BrDU-FITC and propridium iodide. Thesamples were then run on the CytoFLEX flow cytometer with positive andnegative controls provided in the kit (Beckman Coulter, Chaska, Minn.,USA).

Apoptosis assays. CART19 or GM-CSF^(k/o) CART19 cells were stimulatedwith PMA/ionomycin, CD19+ cell line NALM6 or CD3/CD28 beads at differenttime points (0 hr, 1 hr, 2 hr, 4 hr, 6 hr) on a 1:1 ratio. Then, cellswere spin and washed with flow buffer, followed by incubation in thedark with the following antibodies: CD3 (SK7) APC-Cy7 (560176,BioLegend, San Diego, Calif.), Annexin V P E (556421, BD Biosciences,San Jose, Calif.), 7-AAD (559925, BD Biosciences, San Jose, Calif.).Then, the expression of Annexin V and 7-AAD was measured via flowcytometry. Three biological replicates of unstimulated and stimulatedCART19 and GM-CSF^(ko) CART19 cells were included. For assays includingblocking antibodies, TRAIL-R2 (DR5) Fc (10140-T2-100, R&D Systems,Minneapolis, Minn.) and Fas Fc (326-FS-050, R&D Systems, Minneapolis,Minn.) were added at a dose of 10 ng/mL.

Western blot. For immunoblot assays, irradiated target cell line NALM6was co-cultured at a 1:1 ratio with CART19 or GM-CSF^(k/o) CART19 cellsat different time points (0 hr, 2 hr, 4 hr, 6 hr). Cell pellets werewashed with PBS and lysed in 100 uL of RIPA buffer (89900, ThermoFisher, Waltham, Mass., USA), and protein concentration was measured byBCA protein assay (23255, Thermo Fisher, Waltham, Mass., USA). SDS-PAGEgels were used to resolve 30 ug cell lysates, and proteins weretransferred to Nitrocellulose membranes via wet transfer. Nitrocellulosemembranes were blocked with 5% BSA in TBST for 1 hr at room temperature.Membranes were incubated overnight at 4° C. with the followingantibodies: Rabbit BID (2002, Cell Signaling, Danvers, Mass., USA)(dilution 1:1000) and Rabbit β-Actin D6A8 (8457, Cell Signaling,Danvers, Mass., USA). Membranes were washed with TBST and incubated withHRP-conjugated secondary antibodies at a dilution of 1:1000 for 1 hr atroom temperature. Blots were revealed using the SuperSignal West PicoPlus Chemiluminescent substrate (34579, Thermo Fisher, Waltham, Mass.,USA).

Animal Models. 6-8 week old non-obese diabetic/severe combinedimmunodeficient mice bearing a targeted mutation in the interleukin(IL)-2 receptor gamma chain gene (NSG) mice were purchased from JacksonLaboratories (Jackson Laboratories, Bar Harbor, Me., USA) and thenmaintained at the Mayo Clinic animal facility. All animal experimentswere performed under an IACUC approved protocol (A00001767). Mice weremaintained in an animal barrier space that is approved by the IBC forBSL2+ level experiments (IBC #HIP00000252.20). Mice were intravenouslyinjected with 1.0×10⁶ luciferase⁺ JeKo-1 cells. Fourteen days afterinjection, mice were imaged with a bioluminescent imager using an IVIS®Lumina S5 Imaging System (PerkinElmer, Hopkinton, Mass., USA) to confirmengraftment. Imaging was performed 10 minutes after the intraperitonealinjection of 10 μL/g D-luciferin (15 mg/mL, Gold Biotechnology, St.Louis, Mo., USA). Mice were then randomized based on theirbioluminescence imaging to receive different treatments as outlined inthe separate specific experiments. Mice were euthanized for necropsywhen moribund.

Results

Knock out of GM-CSF in CART19 cells enhances their antigen specificproliferation. In order to generate GM-CSF^(k/o) CART cells, CRISPR/Cas9was used to disrupt GM-CSF (CSF2) in CART19 cells, and generated CARTcells that produced little to no GM-CSF upon activation through theirCAR (FIGS. 9 and 18A-18B, see method section, CART Cells, FIG. 24A).GM-CSF disruption in CART cells did not affect the transductionefficiency of T cells (FIG. 18C), change the composition of CART cellproduct (CD4:CD8 ratio) at rest or upon activation (FIG. 18D), or alterCART cell antigen specific killing (FIG. 24B). However, GM-CSF^(k/o)CART19 cells exhibited superior antigen specific proliferation comparedto GM-CSF^(wt) CART19 when stimulated through the CAR19 via a co-culturewith the irradiated CD19+NALM6 cells (FIG. 18D). Importantly, whileantigen specific proliferation of GM-CSF^(k/o) CART19 and GM-CSF^(wt)CART19 was initially similar, it significantly improved after 5 daysfollowing the initial stimulation (FIG. 18E).

GM-CSF editing of CART19 cells is precise and efficient. Having shownthat GM-CSF^(k/o) CART19 cells exhibit enhanced antigen specificproliferation, the aim was to rule out an off-target effect of the CSF2directed gRNA. Whole exome sequencing of CART19 and GM-CSF^(k/o) CART19cells were performed. Using CRISPR/Cas9 to disrupt CSF2 resulted in anefficiency of 60-70% (FIG. 9). Whole exome sequencing (WES) of themodified cells showed no significant difference in SNV or indels betweenGM-CSF^(k/o) and control (GM-CSF^(wt)) CART19 cells (FIG. 19A). WES wasonly significant for alterations in the intended gene target (FIGS.19B-19C). The high precision and specificity of targeting GM-CSF exon 3indicated that improved CART function is unlikely due to an off-targeteffect of the guide RNA, suggesting a direct effect of GM-CSF depletionon CART cells.

Activated T cells and CART19 cells express high levels of GM-CSFreceptors. In order to determine if GM-CSF directly interacts withGM-CSF producing CART cells, whether CART cells express GM-CSF receptors(GM-CSFR) was first studied. While resting CART19 cells do not expressany GM-CSF receptors, the experiments indicated that activated CARTcells significantly upregulate both GM-CSF receptor (GM-CSFR) a and Psubunits. This finding was significant when T cells or CART19 cells werenon-specifically activated through their T cell receptors (FIGS.19D-19F). Additionally, both GM-CSF^(k/o) and GM-CSF^(wt) CART19 cellsupregulated GM-CSFRα and GM-CSFRβ when activated through the CAR withirradiated CD19′ NALM6 cells (FIG. 19G).

GM-CSF knockout in CART cells ameliorate CART cell apoptosis. Next, theaim was to determine if CART cells undergo apoptosis and whether GM-CSFdisruption ameliorates CART cell apoptosis. The expression of Annexin Vand 7-AAD was first measured by flow cytometry at early activation timepoints following either their stimulation through the CAR (through aco-culture with irradiated CD19⁺ NALM6 cells), non-specific stimulationthrough the TCR (CD3/CD28 beads), or non-specific stimulation withPMA/ionomycin. There were significantly more apoptotic CART19 cells(AnnexinV⁺7-AAD−) when stimulated via the CAR or PMA/ionomycin comparedto non-specific stimulation through the TCR (FIGS. 20A-20B).GM-CSF^(k/o) CART19 cells exhibited significantly less apoptosis uponantigen specific stimulation compared to GM-CSF^(wt) CART19 at earlytime points following antigen activation (FIG. 20C). GM-CSF^(k/o) CARTcells also exhibited less apoptosis compared to GM-CSF^(wt) CART cellsupon PMA/ionomycin stimulation (FIG. 20D). To further validate thesefindings, the TUNEL assay was performed, which preferentiallyincorporates BrdU into apoptotic cells. The expression of BrdU on the Tcells in the G0-G1 phase was measured and again found fewer apoptoticCART cells upon antigen-specific stimulation of GM-CSF^(k/o) CART19compared to GM-CSF^(wt) CART19 cells (FIGS. 20E-20F).

GM-CSF producing CART19 cells are intrinsically more susceptible toapoptosis. Since the data indicate that GM-CSF disruption in CART19cells ameliorate their apoptosis upon antigen specific stimulation, andthat activated CART19 cells upregulate GM-CSF receptors, the aim was toinvestigate the mechanisms of this effect. First, the susceptibility toapoptosis of GM-CSF^(k/o) CART cells compared to GM-CSF^(wt) CART cellswas investigated by studying activation pathways in resting CART cells(at the end of CART cell manufacturing (see Methods). Transcriptomeinterrogation of resting GM-CSF^(k/o) CART19 revealed a distinct geneexpression signature compared to resting GM-CSF^(wt) CART19 (FIG. 21A).There are more genes significantly downregulated in the GM-CSF^(k/o)CART19 compared to GM-CSF^(wt) CART19 (FIGS. 21B-21C). The transcriptomepattern of GM-CSF k/o CART19 more closely resembled untransduced T cellsfor wt CART19. Gene set enrichment analysis demonstrated that apoptoticpathways were most significantly altered after GM-CSF disruption inCART19 cells (FIG. 21D). These results indicated that GM-CSF^(k/o) CARTsare less susceptible to apoptosis at their baseline, independent ofsubsequent activation. Next, the aim was to determine whether thereduced apoptosis in GM-CSF^(k/o) CART19 cells is a direct result ofGM-CSF disruption or whether it is due to interactions between secretedGM-CSF and upregulated GM-CSF receptors on CART19 cells. To test this,CART19 cells were expanded in the presence of the GM-CSF neutralizingantibody lenzilumab (see FIG. 24C Schema of CART19 production in thepresence of GM-CSF blocking antibody, [FIG. 21E and FIG. 24C). Followinggeneration of CART19 cells in the presence of GM-CSF neutralizingantibody, their apoptosis was measured after antigen specificstimulation. There was no difference in apoptotic cells (Annexin+7AAD−)between CART19 generated in the presence of lenzilumab or antibodycontrols (FIG. 21E), indicating that amelioration of apoptosis is notdue to interaction between GM-CSF and its upregulated receptors onactivated CART19 cells.

GM-CSF disruption of CART19 cells primes their activation and anti-tumoreffect. Having demonstrated that GM-CSF^(k/o) CART19 cells exhibit lessapoptosis upon antigen specific stimulation, the next aim was to studyhow GM-CSF disruption impacts the level of CART cell activation and howthis impact their proliferation and antitumor activity. Twenty-fourhours following antigen specific stimulation, GM-CSF^(k/o) CART19expressed lower levels of CD3, CD45, CD69, HLA-DR, and CD25 (FIGS.22A-22H), compared to GM-CSF^(wt) CART19 indicating reduced levels of Tcells activation. Then a xenograft model for relapsed lymphoma (FIG.22I) was used to study the impact of GM-CSF knockout of CART19 in vivo.Here, NSG mice were engrafted with JeKo-1 and randomized to receivecontrol T cells, GM-CSF^(k/o) or GM-CSF^(wt) CART19. GM-CSF^(k/o) CART19cells exhibited reduced activation, but enhanced delayed proliferation,and improved antitumor activity after 13 days of treatment and showimproved percent survival at 40 days compared to control T cells similatto that of GM-CSF^(wt) CART19 (FIGS. 22J-22M).

Finally, to determine the mechanisms of reduced AICD following GM-CSFdisruption, the intrinsic and extrinsic regulators of apoptosis wereinterrogated. There was no change in GM-CSF^(k/o) CART cell apoptosisfollowing blockade of extrinsic death pathways using TRAIL:TRAILR orFas:FasL blockade (FIGS. 23A and 23B); however, there was a consistentreduction in Bid following GM-CSF disruption (FIGS. 23C-23D).

Discussion

In this Example a novel mechanism by which GM-CSF affects CART cellfunction is described. It was identified for the first time thatactivated CART cells upregulate GM-CSF receptors, and that GM-CSFdisruption in CART cells ameliorates their apoptosis and AICD, which inturns enhances their antitumor activity. It was demonstrates that GM-CSFmediated CART cell apoptosis is likely a result of cross talk betweenGM-CSF and intrinsic pathways of apoptosis.

Preclinical studies and correlative science from CART19 clinical trialshave shown that inhibitory myeloid cells and cytokines are major playersin both inducing CART cell toxicity and limiting anti-tumor effects.While efforts are predominantly focused on mechanisms of myeloid cellpolarization and interactions with CART cells, no direct interactionbetween myeloid-derived cytokines and CART cells has yet beenidentified. In this study, a novel mechanism is reported by which GM-CSFdirectly affects CART cell function by promoting their apoptosis andAICD.

The present findings have significant therapeutic implications. It hasbecome increasingly evident that CART cells are susceptible to apoptosisand AICD. CART cells upregulate Fas and its ligand (FasL), TRAIL, andTRAIL-R and are prone to FAS- and TRAIL-mediated death when a thresholdof cell activation is reached. The interaction between Fas-FasL withinthe CART cells and tumor microenvironment limits both their persistenceand anti-tumor efficacy, and genetic engineering of the CAR to include aFas dominant negative receptor enhanced anti-tumor activity andpersistence in solid tumor models. Most significantly, a recent studyshowed that TRAIL-deficient CART19 completely lose theirantigen-specific killing abilities. As reports continue to demonstrateclinical safety and feasibility of CRISPR-modified CART cells, thepresent study validates CRISPR/Cas9 GM-CSF^(k/o) CART19 as a potentialnext generation strategy to be tested in B cell malignancies. The datapresented in this this Example suggest a significant advantage for usinggenetic knock out of GM-CSF, which results in amelioration of apoptosisand AICD, in addition to reduction of GM-CSF levels, monocyteactivation, and CART cell toxicities.

In conclusion, the present study reveals that GM-CSF producing CART19cells are more prone to apoptosis and that GM-CSF gene expressiondisruption of CART19 cells reduces their apoptosis and AICD and enhancestheir proliferation and antitumor effect. These findings uncover a newmechanism of reduction in efficacy of CART cell therapy and importantlyilluminates a new avenue to overcome CART cell apoptosis through thedisruption of GM-CSF gene or GM-CSF production.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for treating or preventing CAR-T cellrelated toxicity in a subject in need thereof, the method comprisingadministering to the subject CAR-T cells having a GM-CSF geneinactivation, GM-CSF gene knock-down or gene knockout (GM-CSF^(k/o)CAR-T cells).
 2. The method of claim 1, wherein the CAR-T cell relatedtoxicity comprises neurotoxicity, cytokine release syndrome (CRS) or acombination thereof.
 3. The method of claim 1, wherein the subject has acancer and/or a tumor.
 4. The method of claim 3, wherein the cancer islymphoma or a leukemia.
 5. The method of claim 4, wherein the lymphomais a diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma, orfollicular lymphoma.
 6. The method of claim 4, wherein the leukemia isacute lymphoblastic leukemia (ALL).
 7. The method of claim 3, whereinthe cancer is multiple myeloma.
 8. The method of claim 1, wherein theGM-CSF^(k/o) CAR-T cells target tumor antigen CD19 on lymphoma orleukemia cancer cells.
 9. The method of claim 1, wherein theGM-CSF^(k/o) CAR-T cells target tumor antigen BCMA on multiple myelomacells.
 10. The method of claim 3, wherein levels of the CAR-T cellshaving a GM-CSF gene inactivation, GM-CSF gene knock-down or geneknockout (GM-CSF^(k/o) CAR-T cells) expand and persist in blood of thesubject from a peak level of GM-CSF^(k/o) CAR-T cell expansion duringthe first 30 days after administration of the GM-CSF^(k/o) CAR-T cellsand expansion of the GM-CSF^(k/o) CAR-T cells up to at least 90 days to180 days after the administration of the GM-CSF^(k/o) CAR-T cells. 11.The method of claim 10, wherein GM-CSF^(k/o) CAR-T cell expansion andpersistence in the blood of the subject continues for up to 24 monthsafter administration of the GM-CSF^(k/o) CAR-T cells.
 12. The method ofclaim 10, wherein GM-CSF^(k/o) CAR-T cell expansion and persistence inthe blood of the subject achieves an anti-cancer or anti-tumor efficacyfrom 90 days to 24 months after administration of the GM-CSF^(k/o) CAR-Tcells.
 13. The method of claim 10, wherein GM-CSF^(k/o) CAR-T cells peakexpansion is enhanced relative to wild type (wt) CAR-T cells andpersistence as measured by CAR area under the curve (AUC) is improvedrelative to wt CAR-T cells.
 14. The method of claim 13, wherein improvedGM-CSF^(k/o) CAR-T cell expansion and persistence results in improvedobjective response rates (ORR), improved progression free survival(PFS), or improved overall survival (OS) compared to wt CAR-T cells. 15.The method of claim 12, wherein the anti-cancer or anti-tumor efficacyin the subject is a complete or partial remission of the cancer and/orthe tumor.
 16. The method of claim 12, wherein the anti-cancer oranti-tumor efficacy in the subject is a reduction or an absence of signsand symptoms of the cancer and/or the tumor.
 17. A method for increasingCAR-T cell proliferation in a subject treated with GM-CSF-inactivated orGM-CSF^(k/o) CAR-T cells, the method comprising administering to thesubject CAR-T cells having a GM-CSF gene inactivation, GM-CSF geneknock-down or gene knockout (GM-CSF^(k/o) CAR-T cells), whereinadministration of the GM-CSF^(k/o) CAR-T cells increases CAR-Tproliferation in the subject.
 18. The method of claim 17, whereinadministration of the GM-CSF^(k/o) CAR-T cells and expansion of theGM-CSF^(k/o) CAR-T cells reduces production of GM-CSF by 75%-99% oreliminates production of GM-CSF by the GM-CSF^(k/o) CAR-T cells.
 19. Themethod of claim 18, wherein reduction or elimination of the productionof GM-CSF by the GM-CSF^(k/o) CAR-T cells increases production andexpansion of the GM-CSF by the GM-CSF^(k/o) CAR-T cells.
 20. The methodof claim 19, wherein increased production and expansion of the GM-CSF bythe GM-CSF^(k/o) CAR-T cells reduces of eliminates CAR-T cell relatedtoxicity in the subject, wherein the CAR-T cell related toxicitycomprises neurotoxicity, cytokine release syndrome (CRS) or acombination thereof.
 21. The method of claim 17, wherein the subject hasa cancer and/or a tumor.
 22. The method of claim 21, wherein the canceris lymphoma or a leukemia.
 23. The method of claim 22, wherein thelymphoma is a diffuse large B cell lymphoma (DLBCL).
 24. The method ofclaim 22, wherein the leukemia is acute lymphoblastic leukemia (ALL).25. The method of claim 21, wherein the cancer is multiple myeloma. 26.The method of claim 17, wherein the GM-CSF^(k/o) CAR-T cells targettumor antigen CD19 on lymphoma or leukemia cancer cells.
 27. The methodof claim 17, wherein the GM-CSF^(k/o) CAR-T cells target tumor antigenBCMA on multiple myeloma cells.
 28. The method of claim 18, whereinlevels of the CAR-T cells having a GM-CSF gene inactivation, GM-CSF geneknock-down or gene knockout (GM-CSF^(k/o) CAR-T cells) expand andpersist in blood of the subject from a peak level of GM-CSF^(k/o) CAR-Tcell expansion during the first 30 days after administration of theGM-CSF^(k/o) CAR-T cells and expansion of the GM-CSF^(k/o) CAR-T cellsup to at least 90 days to 180 days after the administration of theGM-CSF^(k/o) CAR-T cells.
 29. The method of claim 28, whereinGM-CSF^(k/o) CAR-T cell expansion and persistence in the blood of thesubject continues for up to 24 months after administration of theGM-CSF^(k/o) CAR-T cells.
 30. The method of claim 28, whereinGM-CSF^(k/o) CAR-T cell expansion and persistence in the blood of thesubject achieves an anti-cancer or anti-tumor efficacy from 90 days to24 months after administration of the GM-CSF^(k/o) CAR-T cells.
 31. Themethod of claim 28, wherein GM-CSF^(k/o) CAR-T cells peak expansion isenhanced relative to wt CAR-T cells and persistence as measured by CARarea under the curve (AUC) is improved relative to wt CAR-T cells. 32.The method of claim 31, wherein improved GM-CSF^(k/o) CAR-T cellexpansion and persistence results in improved objective response rates(ORR), improved progression free survival (PFS), or improved overallsurvival (OS) compared to wt CAR-T cells.
 33. The method of claim 30,wherein the anti-cancer or anti-tumor efficacy in the subject is acomplete or partial remission of the cancer and/or the tumor.
 34. Themethod of claim 30, wherein the anti-cancer or anti-tumor efficacy inthe subject is a reduction or an absence of signs and symptoms of thecancer and/or the tumor.